U.S. patent application number 13/527210 was filed with the patent office on 2014-08-28 for ultra-sensitive detection of molecules on single molecule arrays.
This patent application is currently assigned to Quanterix Corporation. The applicant listed for this patent is David C. Duffy, Evan Ferrell, Jeffrey D. Randall, David M. Rissin, David R. Walt. Invention is credited to David C. Duffy, Evan Ferrell, Jeffrey D. Randall, David M. Rissin, David R. Walt.
Application Number | 20140243223 13/527210 |
Document ID | / |
Family ID | 42038065 |
Filed Date | 2014-08-28 |
United States Patent
Application |
20140243223 |
Kind Code |
A1 |
Duffy; David C. ; et
al. |
August 28, 2014 |
ULTRA-SENSITIVE DETECTION OF MOLECULES ON SINGLE MOLECULE
ARRAYS
Abstract
The present invention relates to systems and methods for
detecting analyte molecules or particles in a fluid sample and in
some cases, determining a measure of the concentration of the
molecules or particles in the fluid sample. Methods of the present
invention may comprise immobilizing a plurality of analyte
molecules or particles to form a plurality of complexes, releasing
at least a portion of some of the plurality of complexes,
determining at least a portion of the plurality of complexes
released, and determining a measure of the concentration of the
analyte molecules or particles in a fluid sample.
Inventors: |
Duffy; David C.;
(Somerville, MA) ; Ferrell; Evan; (Brighton,
MA) ; Randall; Jeffrey D.; (Canton, MA) ;
Rissin; David M.; (Somerville, MA) ; Walt; David
R.; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Duffy; David C.
Ferrell; Evan
Randall; Jeffrey D.
Rissin; David M.
Walt; David R. |
Somerville
Brighton
Canton
Somerville
Boston |
MA
MA
MA
MA
MA |
US
US
US
US
US |
|
|
Assignee: |
Quanterix Corporation
Lexington
MA
|
Family ID: |
42038065 |
Appl. No.: |
13/527210 |
Filed: |
June 19, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12236486 |
Sep 23, 2008 |
8222047 |
|
|
13527210 |
|
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|
Current U.S.
Class: |
506/9 ; 435/6.11;
435/7.92; 436/501; 436/94 |
Current CPC
Class: |
G01N 33/54386 20130101;
G01N 33/54313 20130101; G01N 33/54326 20130101; C12Q 1/6825
20130101; Y10T 436/143333 20150115 |
Class at
Publication: |
506/9 ; 436/501;
435/7.92; 436/94; 435/6.11 |
International
Class: |
G01N 33/543 20060101
G01N033/543; C12Q 1/68 20060101 C12Q001/68 |
Claims
1-199. (canceled)
200. A method of quantifying a molecular species, comprising:
providing a plurality of particles, wherein at least some of
particles are associated with at least one molecule of the
molecular species; partitioning at least a portion of the plurality
of particles across a plurality of reaction vessels so that a
statistically significant fraction of the reaction vessels contain
no particles associated with a molecule of the molecular species
and a statistically significant fraction of reaction vessels
contain at least one particle associated with at least one molecule
of the molecular species; detecting the presence or absence of a
molecule of the molecular species in at least some of the reaction
vessels and determining the percentage of reaction vessels which
contain a molecule of the molecular species; and determining a
measure of the quantity of the molecular species based on the
percentage of reaction vessels which contain a molecule of the
molecular species.
201. The method of claim 200, wherein either one or no molecules of
the molecular species to be detected are immobilized with respect
to a single particle.
202. The method of claim 200, further comprising an act of
determining the concentration of molecular species in the
fluid.
203. The method of claim 200, wherein each of the particles
comprise a plurality of binding partners for the molecular
species.
204. The method of claim 200, wherein the volume of each of the
plurality of reaction vessels is between about 10 attoliters and
about 100 picoliters.
205. The method of claim 200, wherein the volume of each of the
plurality of reaction vessels is between about 1 femtoliter and
about 1 picoliter.
206. The method of claim 200, wherein the volume of each of the
plurality of reaction vessels is between about 10 femtoliters and
about 100 femtoliters.
207. The method of claim 200, wherein the presence or absence of a
particle associated with at least one molecule of the molecular
species is detected directly.
208. The method of claim 200, wherein the presence or absence of a
reaction vessel containing a particle associated with at least one
molecule of the molecular species is detected indirectly.
209. The method of claim 208, wherein each of the plurality of
reaction vessels is exposed to at least one precursor labeling
agent molecule.
210. The method of claim 209, wherein the at least one precursor
labeling agent molecule is converted to a labeling agent molecule
when contained in a reaction vessel containing a particle
associated with at least one molecule of the molecular species.
211. The method of claim 210, wherein the presence or absence of a
molecule of the molecular species in a reaction vessels is detected
by detecting the presence or absence of a labeling agent molecule
in the reaction vessel.
212. The method of claim 200, wherein the molecular species are
proteins.
213. The method of claim 200, wherein the molecular species are
nucleic acids.
214. The method of claim 200, wherein the molecular species are
small molecules.
215. A method of detecting a molecular species in a fluid sample,
comprising: exposing the fluid sample to a plurality of particles,
wherein at least some of the particles associate with molecules of
the molecular species; partitioning at least a portion of the
plurality of particles across a plurality of reaction vessels,
wherein the plurality of particles are partitioned such that a
statistically significant fraction of the reaction vessels contain
no particles associated with a molecule of the molecular species
and a statistically significant fraction of reaction vessels
contain at least one particle associated with at least one molecule
of the molecular species to be detected; and determining the
presence or absence of a molecule of the molecular species in at
least some of the reaction vessels.
216. The method of claim 200, wherein the plurality of particles
are partitioned such that a statistically significant fraction of
the reaction vessels contain no particles associated with a
molecule of the molecular species and a statistically significant
fraction of reaction vessels contain one particle associated with
one molecule of the molecular species.
217. The method of claim 200, further comprising determining the
number of the plurality of reaction vessels and/or fraction of the
plurality of reaction vessels that contain or do not contain a
particle associated with a molecule of the molecular species.
218. The method of claim 217, wherein the number or fraction of the
plurality of reaction vessels that contain a particle associated
with a molecule of the molecular species is related to the
concentration of species in the fluid.
219. The method of claim 217, wherein the number or fraction of the
plurality of reaction vessels that contain a particle associated
with a molecule of the molecular species is directly proportional
to the concentration of molecular species in the fluid.
220. The method of claim 215, wherein each of the plurality of
particles comprises a plurality of binding partners for the
molecular species.
221. The method of claim 215, wherein the volume of each of the
plurality of reaction vessels is between about 10 attoliters and
about 100 picoliters.
222. The method of claim 215, wherein the volume of each of the
plurality of reaction vessels is between about 10 femtoliters and
about 100 femtoliters.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/236,486, filed Sep. 23, 2008, entitled
"ULTRA-SENSITIVE DETECTION OF MOLECULES ON SINGLE MOLECULE ARRAYS,"
which is incorporated herein by reference in its entirety for all
purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to systems and methods for
detecting analyte molecules or particles in a fluid sample and in
some cases, determining a measure of the concentration of the
molecules or particles in the fluid sample.
BACKGROUND OF THE INVENTION
[0003] Methods and systems that are able to quickly and accurately
detect and, in certain cases, quantify a target analyte molecule or
particle in a sample are the cornerstones of modern analytical
measurements. Such systems and/or methods are employed in many
areas such as academic and industrial research, environmental
assessment, food safety, medical diagnosis, and detection of
chemical, biological and/or radiological warfare agents.
Advantageous features of such techniques may include specificity,
speed, and sensitivity.
[0004] Most current techniques for quantifying low levels of
analyte molecules in a sample use amplification procedures to
increase the number of reporter molecules in order to be able to
provide a measurable signal. For example, these known processes
include enzyme-linked immunosorbent assays (ELISA) for amplifying
the signal in antibody-based assays, as well as the polymerase
chain reaction (PCR) for amplifying target DNA strands in DNA-based
assays. A more sensitive but indirect protein target amplification
technique, called immunoPCR (see Sano, T.; Smith, C. L.; Cantor, C.
R. Science 1992, 258, 120-122), makes use of oligonucleotide
markers, which can subsequently be amplified using PCR and detected
using a DNA hybridization assay (see Nam, J. M.; Thaxton, C. S.;
Mirkin, C. A. Science 2003; 301, 1884-1886; Niemeyer, C. M.; Adler,
M.; Pignataro, B.; Lenhert, S.; Gao, S.; Chi, L. F.; Fuchs, H.;
Blohm, D. Nucleic Acids Research 1999, 27, 4553-4561; and Zhou, H.;
Fisher, R. J.; Papas, T. S, Nucleic Acids Research 1993, 21,
6038-6039). While the immuno-PCR method permits ultra low-level
protein detection, it is a complex assay procedure, and can be
prone to false-positive signal generation (see Niemeyer, C. M.;
Adler, M.; Wacker, R. Trends in Biotechnology 2005, 23,
208-216).
[0005] One disadvantage of typical known methods and/or systems for
accurately detecting and, optionally, quantifying low
concentrations of a particular analyte in solution is that they are
based on ensemble responses in which many analyte molecules give
rise to the measured signal. Most detection schemes require that a
large number of molecules are present in the ensemble for the
aggregate signal to be above the detection threshold. This
disadvantage limits the sensitivity of most detection techniques
and the dynamic range (i.e., the range of concentrations that can
be detected). Many of the known methods and techniques are further
plagued with problems of non-specific binding, which is the binding
of analyte molecules/particles to be detected or reporter species
non-specifically to sites other than those expected. This leads to
an increase in the background signal, and therefore limits the
lowest concentration that may be accurately or reproducibly
detected.
[0006] Accordingly, improved methods for detecting and, optionally,
quantifying analyte molecules or particles, especially in samples
where such molecules or particles are present at very low
concentration are needed.
SUMMARY OF THE INVENTION
[0007] The present invention relates to systems and methods for
detecting analyte molecules or particles in a fluid sample and in
some cases, determining a measure of the concentration of the
molecules or particles in the fluid sample. The subject matter of
the present invention involves, in some cases, interrelated
products, alternative solutions to a particular problem, and/or a
plurality of different uses of one or more systems and/or
articles.
[0008] In one aspect, the invention is directed towards a method.
According to one set of embodiments, a method of detecting analyte
molecules or particles in a fluid sample, comprises exposing a
substrate comprising a plurality of capture components to a sample
comprising a plurality of analyte molecules or particles, so that
analyte molecules or particles associate with capture components to
form a plurality of complexes, each complex being immobilized with
respect to the substrate and comprising at least one capture
component and at least one analyte molecule or particle,
dissociating at least a portion of each complex to form a plurality
of dissociated species, which are not immobilized with respect to
the substrate, partitioning the plurality of dissociated species
across a plurality of reaction vessels, and determining the
presence or absence of a dissociated species in at least one
reaction vessel.
[0009] In one aspect, the invention is directed towards a system.
According to one set of embodiments, a system for detecting analyte
molecules or particles in a fluid sample, comprises a first
substrate comprising a plurality of capture components able to
become specifically immobilized with respect to a plurality of
analyte molecules or particles of a sample, such that the analyte
molecules or particles associate with capture components to form a
plurality of complexes, wherein the complexes are susceptible to
being dissociated from the first substrate by a dissociating agent
to form a plurality of dissociated species, and a second substrate
comprising an array comprising a plurality of reaction vessels, the
reaction vessels being configured to receive and contain
dissociated species therein.
[0010] According to another set of embodiments, a method for
determining a measure of the concentration of analyte molecules or
particles in a fluid sample, comprises capturing a plurality of
analyte molecules or particles on a first substrate, releasing a
plurality of molecules or particles from the first substrate,
detecting molecules or particles released from the first substrate
on or within a second substrate comprising a plurality of reaction
vessels, and determining a measure of the concentration of the
analyte molecules or particles in the fluid sample based on the
detection of molecules or particles released from the first
substrate on or within the second substrate.
[0011] According to another set of embodiments, a method for
determining a measure of the concentration of the analyte molecules
or particles in a fluid sample, comprises capturing a plurality of
analyte molecules or particles on a first substrate, releasing a
plurality of molecules or particles from the first substrate,
detecting molecules or particles released from the first substrate
on or within a second substrate, and determining a measure of the
concentration of the analyte molecules or particles in the fluid
sample based on the detection of molecules or particles released
from the first substrate, wherein the true concentration of the
analyte molecules or particles in the fluid sample is less than
about 50.times.10.sup.-15 molar, and wherein the measure of the
concentration determined in the determining act differs from the
true concentration by no greater than 10%.
[0012] According to yet another set of embodiments, a method for
determining a measure of the concentration of analyte molecules or
particles in a fluid sample, comprises capturing a plurality of
analyte molecules or particles on a first substrate, releasing a
plurality of molecules or particles from the first substrate,
immobilizing at least one particle comprising a binding partner for
the molecule or particle released from the first substrate to each
of at least a fraction of the plurality of molecules or particles
released from the first substrate to form a plurality of complexes,
detecting the plurality of complexes formed in the immobilizing
act, and determining a measure of the concentration of the analyte
molecules or particles in the fluid sample based on the detection
of the complexes.
[0013] In some embodiments, a method for determining a measure of
the concentration of analyte molecules or particles in a fluid
sample, comprises, capturing a plurality of analyte molecules or
particles on a first substrate by immobilizing the plurality of
analyte molecules or particles on the first substrate, releasing a
plurality of immobilized molecules or particles from the first
substrate, wherein the number of molecules or particles released is
related to the number of analyte molecules or particles captured,
detecting molecules or particles released from the first substrate
on or within a second substrate, wherein at least a fraction of the
molecules or particles released from the first substrate become
immobilized with respect to the second substrate, and wherein the
number of molecules or particles detected on or within the second
substrate is no greater than the number of molecules or particles
which are released from the first substrate, and determining a
measure of the concentration of the analyte molecules or particles
in the fluid sample based on the detection of molecules or
particles released from the first substrate.
[0014] In another set of embodiments, a method for determining a
measure of the concentration of analyte molecules or particles in a
fluid sample, comprises capturing a plurality of analyte molecules
or particles on a first substrate by immobilizing the plurality of
analyte molecules or particles on the first substrate, releasing a
plurality of immobilized molecules or particles from the first
substrate, wherein the number of molecules or particles released is
related to the number of analyte molecules or particles captured,
immobilizing at least one first binding ligand with respect to each
of at least a fraction of the plurality of analyte molecules or
particles or to each of at least a fraction of the plurality of
molecules or particles released from the first substrate, capturing
at least a fraction of the plurality of molecules or particles
released from the first substrate on or within a second substrate,
exposing the second substrate to a plurality of precursor labeling
agent molecules, wherein the plurality of precursor labeling agent
molecules are converted to a plurality of labeling agent molecules
upon exposure to first binding ligands, and determining a measure
of the concentration of the analyte molecules or particles in the
fluid sample based on the detection of the labeling agent
molecules.
[0015] In still yet another set of embodiments, a method of
detecting analyte molecules or particles in a fluid sample,
comprises exposing a substrate comprising a plurality of capture
components to a sample comprising a plurality of analyte molecules
or particles, so that analyte molecules or particles associate with
capture components to form a plurality of complexes, each complex
being immobilized with respect to the substrate and comprising at
least one capture component and at least one analyte molecule or
particle, dissociating at least a portion of each complex to form a
plurality of dissociated species, which are not immobilized with
respect to the substrate, and detecting at least some of the
plurality of the dissociated species substantially simultaneously,
wherein substantially all of the detected dissociated species are
spatially separated with respect to the other detected dissociated
species during detection, such that detection is able to resolve
individual dissociated species of the plurality of dissociated
species.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic flow diagram depicting one embodiment
of steps (A-E) for performing one method of the present
invention;
[0017] FIG. 2 is a schematic flow diagram depicting a sequence of
steps (A-E) of one embodiment of one method of the present
invention;
[0018] FIG. 3 is a schematic flow diagram depicting embodiments
(A-C) of how an immobilized complex may dissociate from a substrate
according to certain embodiments;
[0019] FIG. 4 is a schematic flow diagram illustrating immobilized
complexes comprising cleavable linkages and cleaving of the
cleavable linkages, according to various embodiments (A-C);
[0020] FIG. 5 is a schematic flow diagram depicting steps (A-E) of
one embodiment of one method of the present invention;
[0021] FIG. 6 depicts a second substrate comprising a plurality of
capture components with which a plurality of dissociated species
may become associated as shown by steps (A-B), according to one
embodiment;
[0022] FIG. 7 is a schematic flow diagram depicting steps (A-E) of
one embodiment of one method of the present invention;
[0023] FIG. 8 is a schematic flow diagram depicting steps (A-E) of
one method of the present invention, according to one
embodiment;
[0024] FIG. 9 is a schematic flow diagram depicting steps (A-D) of
one embodiment of one method of the present invention;
[0025] FIGS. 10A-10C show non-limiting examples of substrates that
may be used according to certain embodiments of the present
invention;
[0026] FIG. 11 is a schematic flow diagram depicting an embodiment
of a method (steps A-D) for the formation of a plurality of
reaction vessels through mating of a substrate and a sealing
component and depicting examples of the size (E, F) of a sealing
component relative to a substrate;
[0027] FIG. 12 shows side view cross-section schematics showing an
etched fiber optic bundle that forms an array of microwells (steps
A-D), according to one embodiment of the present invention, and
depicting the localization of a capture component within a reaction
vessel (steps E-G), according to some embodiments of the present
invention;
[0028] FIG. 13A is a photocopy of a photomicrograph of an entire
fiber optic array, with an inset showing a close-up of the bundle,
according to one embodiment of the present invention;
[0029] FIG. 13B is a photocopy of an atomic force microscope (AFM)
image of a portion of an etched surface of a fiber optic array,
according to one embodiment of the present invention;
[0030] FIG. 14A depicts an experimental set-up for detection using
light, according to one embodiment of the present invention;
[0031] FIG. 14B shows a fiber optic array that has been sealed with
a sealing component, according to one embodiment;
[0032] FIG. 15A is a schematic flow diagram depicting a method of
indirectly detecting a dissociated species and/or a molecule or
particle released from a substrate, according to some
embodiments;
[0033] FIG. 15B is a schematic flow diagram depicting a method of
indirectly detecting a dissociated species and/or a molecule or
particle released from a substrate, according to some
embodiments;
[0034] FIG. 16 is a schematic flow diagram depicting steps (A-B) of
one embodiment of one method of the present invention in which
dissociated species are immobilized with respect to particles prior
to partitioning across a plurality of reaction vessels;
[0035] FIGS. 17A and 17B show non-limiting examples of a system
employing an optical detection system of the present invention
according to some embodiments;
[0036] FIG. 18 is a schematic block diagram showing a system
employing a fiber optic assembly with an optical detection system
according to an embodiment of the invention;
[0037] FIG. 19 shows a graph of a schematic calibration curve of a
form that may be used to determine the concentration of an analyte
molecule or particle in a fluid sample, according to some
embodiments of the present invention;
[0038] FIG. 20A shows a plot of the percent active wells against
the concentration of TNF-alpha in serum samples, according to one
example of the present invention;
[0039] FIG. 20B shows a magnified view of the lower end of the
graph in FIG. 20A;
[0040] FIG. 21 shows a plot of the percent active wells against
concentration of prostate specific antigen (PSA) in serum samples,
in one embodiment of the present invention;
[0041] FIG. 22 shows a plot of the percent active wells against
concentration of interleukin-6 (IL-6) in serum samples, in one
embodiment of the present invention;
[0042] FIG. 23 shows a plot of the percent active wells against
concentration of vascular endothelial growth factor (VEGF) in serum
samples, in one embodiment of the present invention;
[0043] FIG. 24 is a graph depicting the effects of the
concentration of beta-mercaptoethanol on the release of dissociated
species, in one embodiment of the invention;
[0044] FIG. 25 is a graph depicting the effects of removing
beta-mercaptoethanol before detection of the dissociated species on
an array, in one embodiment of the invention;
[0045] FIG. 26 is a graph depicting results of an example of the
optimization of non-specific binding, according to one method of
the present invention;
[0046] FIG. 27 is a graph depicting results of the detection of
tumor necrosis factor-alpha (TNF-alpha) using a method/system of
the present invention, according to one embodiment;
[0047] FIG. 28 is a graph depicting results of the detection of
TNF-alpha using a standardized ELISA assay;
[0048] FIG. 29 is a graph depicting results of the detection of
TNF-alpha using one embodiment of a method of the present
invention, wherein the first substrate comprised beads and the
beads were modified with antibody (Ab) stabilized with bovine serum
albumin (BSA);
[0049] FIG. 30 is a graph depicting results of an assay involving
the immobilization of Fab' fragments on anti-TNF (MAB610) generated
using enzymatic degradation and purification, according to some
embodiments of the present invention;
[0050] FIG. 31 is a graph depicting of an assay using a 96-well
plate as the first substrate, followed by rebinding of a plurality
of dissociated species to a biotin array, according to one
embodiment of the present invention;
[0051] FIG. 32 is a graph depicting the results of method according
to an embodiment of the present invention, wherein one of the
binding ligands of the assay comprised a photocleavable
linkage;
[0052] FIG. 33 is a graph depicting the detection of TNF-alpha
using a standardized ELISA assay;
[0053] FIG. 34 is a graph depicting the detection of TNF-alpha
using a method/system of the present invention, according to one
embodiment.
[0054] Other aspects, embodiments and features of the invention
will become apparent from the following detailed description when
considered in conjunction with the accompanying drawings. The
accompanying figures are schematic and are not intended to be drawn
to scale. For purposes of clarity, not every component is labeled
in every figure, nor is every component of each embodiment of the
invention shown where illustration is not necessary to allow those
of ordinary skill in the art to understand the invention. All
patent applications and patents incorporated herein by reference
are incorporated by reference in their entirety. In case of
conflict, the present specification, including definitions, will
control.
DETAILED DESCRIPTION
[0055] The present invention relates to systems and methods for the
detection and/or quantification of analyte molecules, particles
(such as, for example, cells, cell organelles and other biological
or non-biological particulates) and the like, in a sample. The
subject matter of the present invention involves, in some cases,
interrelated products, alternative solutions to a particular
problem, and/or a plurality of different uses of one or more
systems and/or articles. It should be understood, that while much
of the discussion below is directed to analyte molecules, this is
by way of example only, and other materials may be detected and/or
quantified, for example, analyte particles. Particular examples of
analytes will be discussed more below.
[0056] The systems and methods of the present invention in certain
instances may help reduce the negative effects of non-specific
binding on detection sensitivity when compared to typical
conventional systems and methods for performing similar assays.
Non-specific binding is the binding or association in a
non-specific fashion of one component of an assay with another
component of the assay with which it is not desirable that it
interact. For example, association, binding, or immobilization of a
binding ligand with the material forming a reaction vessel wall as
opposed to with an analyte molecule or particle to which it has
binding specificity. Non-specific binding may lead to false
positive signals. Non-specific binding may not only affect the
accuracy of the assay measurement, but may also limit the lowest
level of detection. Therefore, methods and/or systems of the
present invention, which in certain embodiments involve
improvements in the level of non-specific binding, may allow for
the detection and/or quantification of analyte molecules in a
sample at a lower detection limit as compared to typical current
technologies. In addition, certain embodiments of the methods
and/or systems of the present invention may also allow for the
detection and/or quantification of analyte molecules in certain
samples that have previously been undetected and/or unquantifiable
in such samples because of the very low concentration in which they
are present.
[0057] "Capture-and-release" systems and methods of certain
embodiments of present invention which utilize a first substrate
and a second substrate also can provide additional benefits as
compared to a basic capture assay that employs only a single
substrate. For example, the first substrate may be configured to
provide a greater surface area, greater volumetric capacity, and/or
increased number/density of capture components as compared to an
assay employing only one substrate. That is, the surface area
and/or volumetric capacity of the first substrate may be greater
than the substrate used in an assay employing only one substrate
such that the analyte molecules may be more effectively, rapidly
and/or thoroughly immobilized with respect to the first substrate
versus immobilization of the target analyte in the sample directly
on a single substrate where detection also occurs. The required
exposure time of the substrate to the analyte molecules may also be
decreased in some cases using an assay of the present invention due
to an increase in surface area and/or volumetric capacity of the
substrate (e.g., by increasing binding kinetics).
[0058] In certain embodiments, the methods for detection and/or
quantifying analyte molecules in a sample comprise a
capture-and-release protocol, wherein the analyte molecules are
captured on a substrate and form a plurality of complexes
associated with the substrate. The substrate may be optionally
exposed to additional reaction components such as at least one
binding ligand, as discussed more below. At least a portion of the
plurality of complexes may be dissociated or otherwise released
from the substrate, for example, by exposing the complexes to a
dissociating agent (e.g., a reducing agent) and/or by cleaving a
cleavable linkage (e.g., a disulfide linkage or a photocleavable
linkage) comprising at least a portion of the complexes
dissociated. In some cases, a dissociable/dissociated complex may
comprise an enzymatic component, which in certain embodiments may
be used to facilitate dissociation and/or detection of the
dissociated complex. At least a portion of the plurality of
dissociated complexes may be detected and may be used to determine
the presence of and/or quantify the analyte molecules in the
sample. The dissociated complexes may be detected either directly
or indirectly, as explained in more detail below.
[0059] An assay method according to some embodiments of the present
invention is illustrated in FIG. 1. A first substrate 200 is
exposed to a sample comprising a plurality of analyte molecules 202
(step (A)). At least a portion of the analyte molecules associate
with the first substrate 200 to form a plurality of immobilized
complexes 204, however, some analyte molecules 206 may not
associate with the first substrate (step (B)). At least a portion
of at least some of the immobilized complexes are dissociated from
the substrate to form a plurality of dissociated species 210 (step
(C)). The dissociated species, in the present example are not
immobilized with respect to the first substrate. In some cases, at
least a portion of the immobilized complexes 208 remain associated
with the first substrate. The plurality of dissociated species 210
are exposed to a second substrate 212 (step (D)), and at least some
of the dissociated species associate with the second substrate 212
(step (E)). The plurality of dissociated species that associate
with the second substrate can then be detected and/or quantified
and, thereby, the analyte molecules in the sample can be detected
and/or quantified.
[0060] Additional components and/or method steps may be utilized in
combination with the above described method. For example, in some
cases, at least one of the first substrate and/or second substrate
may comprise a plurality of capture components with which the
analyte molecules and/or dissociated species may associate. As
another example, in certain embodiments, at least a portion of at
least some of the immobilized complexes dissociate from the
substrate to form a plurality of dissociated species, wherein,
depending on the particular design of the assay as explained below,
the dissociated species may or may not comprise any portion of an
analyte molecule and/or capture component. For example, the
substrate may be additionally exposed to a plurality of at least
one type of binding ligand such that at least one binding ligand
associates with at least a portion of the plurality of complexes,
either before, after or essentially simultaneously with the
immobilization of the complex on the substrate. Such an embodiment
is illustrated in FIG. 2. Substrate 2 comprising a plurality of
capture components 4 (step (A)) is exposed to plurality of analyte
molecules 6, as indicated by arrow 5. Analyte molecules 6 associate
with one of the plurality of capture components 4 (step (B)). The
substrate is then exposed to an optional binding ligand 10, as
indicated by arrow 9, such that a binding ligand associates with at
least a portion of the analyte molecules associated with the
substrate and form a plurality of immobilized complexes 8 (step
(C)). At least a portion of some of the plurality of immobilized
complexes may be dissociated to form a plurality of dissociated
species 12, as indicated by arrow 11 (step (D)). The dissociated
species may or may not comprise a portion of an analyte molecule, a
capture component and/or a binding ligand. In the depicted example,
a portion 12 of each of the binding ligands were dissociated. At
least a portion of the plurality of dissociated species may be
quantified and/or detected, as indicated by arrow 13 (step
(E)).
[0061] In certain embodiments, the invention provides methods for
detecting and/or quantifying a plurality of dissociated species.
The method may comprise partitioning the plurality of dissociated
species across a plurality of reaction vessels, such that at least
some of the reaction vessels contain at least one or, in certain
cases, one dissociated species and at least some of the reaction
vessels contain no dissociated species. In some cases, the
plurality of dissociated species may be partitioned across a
plurality of reaction vessels, such that a statistically
significant fraction of the reaction vessels contains at least one
or, in certain cases, one dissociated species and a statistically
significant fraction of reaction vessels contain no dissociated
species. The presence or absence of a dissociated species in a
reaction vessel may be determined to determine the number of
reaction vessels which contain zero, one, or multiple dissociated
species. The number of reaction vessels containing various
quantities of dissociated species thusly determined may then be
used to quantify the dissociated species, and additionally may be
used to determine a measure of the concentration of the analyte
molecules in the original fluid sample.
[0062] The foregoing methods can be performed using a number of
different assay formats, different reaction conditions, and/or
detection systems in different embodiments of the invention. In
certain embodiments, the reaction vessels may be adapted to capture
the dissociated species of interest. Depending on the assay format,
the reaction vessel may comprise a microwell and a dissociated
species capture component (also referred to as a detection capture
component) for capturing the dissociated species, as discussed more
below.
[0063] In certain embodiments, the present inventions involve
exposing a substrate comprising a plurality of capture components
to a plurality of analyte molecules. A "capture component", as used
herein, is any molecule, other chemical/biological entity or solid
support modification disposed upon a solid support that can be used
to specifically attach, bind or otherwise capture a target molecule
or particle (e.g., an analyte molecule or dissociated species),
such that the target molecule/particle becomes immobilized with
respect to the capture component and solid substrate. As used
herein, "immobilized" means captured, attached, bound, or affixed
so as to prevent dissociation or loss of the target
molecule/particle, but does not require absolute immobility with
respect to either the capture component or the solid substrate.
Capture components which are useful or potentially useful for
practicing certain aspects and embodiments of the invention are
discussed in more detail below. At least some of the analyte
molecules, upon exposure to the substrate comprising a plurality of
capture components, can become immobilized with respect to a
capture component, thereby forming a plurality of immobilized
complexes. For example, in certain embodiments, substantially all
of the plurality of analyte molecules may become immobilized with
respect to capture components such that essentially each of the
plurality of immobilized complexes comprises a capture component
and an analyte molecule.
[0064] The substrate on or in which the plurality of immobilized
complexes is immobilized may comprise a microtiter plate, a
plurality of beads (e.g., magnetic beads), or any other substrate
that, in certain embodiments, comprises a plurality of capture
components, as discussed more below
[0065] In some embodiments, the substrate comprising the
immobilized complexes may be exposed to at least one additional
reaction component, for example, at least a first type of binding
ligand. A "binding ligand," as used herein, is any molecule,
particle, or the like which specifically binds to or otherwise
specifically associates with an analyte molecule, immobilized
complex and/or dissociated species or another molecule or particle
bound to or otherwise associated with the analyte molecule,
immobilized complex and/or dissociated species (e.g., another
binding ligand). In certain embodiments, the binding ligand can
convert a precursor labeling agent molecule to a labeling agent, as
discussed more below. More than one type of binding ligand may be
employed in any given assay method, for example, a first type of
binding ligand and a second type of binding ligand. In one example,
the first binding ligand is able to associate with an analyte
molecule and the second binding ligand is able to associate with
the first binding ligand. When the substrate is exposed to a
plurality of types of binding ligand, at least some of the
plurality of immobilized complexes may additionally comprise, in
some cases, at least one of each type of binding ligand. In certain
embodiments, the binding ligand can be exposed to the substrate
after capture of the analyte molecule so that the binding ligand
binds to the immobilized complex. In other embodiments, the binding
ligand may become associated with the analyte molecule to form a
complex followed by capture of the complex by the substrate to form
the immobilized complex. In yet other embodiments, the binding
ligand may bind to the dissociated species formed upon release of
the immobilized complex, or portion thereof, from the
substrate.
[0066] In some embodiments, a plurality of dissociated species may
be formed by dissociating at least a portion of at least some of
the plurality of immobilized complexes. In certain embodiments, a
plurality of molecules or particles may be released from the
substrate. Any or each of the plurality of dissociated species
(e.g., molecules or particles released from a substrate) may
comprise any portion of an immobilized complex. For example, the
dissociated species may comprise (i) at least a portion of a
binding ligand, (ii) at least a portion of a binding ligand and at
least a portion of the analyte molecule, (iii) at least a portion
of a binding ligand, at least a portion of an analyte molecule and
at least a portion of a capture component, or (iv) essentially all
of the immobilized complex.
[0067] Non-limiting examples of how a dissociated species may be
released from the substrate are depicted in FIG. 3. In scheme (A),
a portion 68 of a binding ligand 64 is dissociated from immobilized
complex 58, which comprises capture component 60, analyte molecule
62, and binding ligand 64. The remainder 66 of binding ligand 64
remains associated with analyte molecule 62. Scheme (B) shows the
formation of a dissociated species 74 comprising a portion 72 of
the analyte molecule 62 and binding ligand 64. The remainder 70 of
the analyte molecule 62 remains associated with capture component
60. Scheme (C) shows the formation of a dissociated species 80
comprising binding ligand 64, analyte molecule 60, and a portion 76
of capture component 60, while the remainder 78 of capture
component 60 remains attached to the substrate. As would be
understood by those skilled in the art given the revelation of this
disclosure, many other possibilities are potentially feasible.
[0068] The plurality of immobilized complexes may be dissociated in
certain embodiments by exposure to a dissociating agent. A
"dissociating agent," as used herein, is any agent that causes at
least a portion of an immobilized complex to dissociate, thereby
forming a dissociated species. For example, the dissociating agent
may be a chemical dissociating agent, electromagnetic radiation, a
change in pH, a change in temperature, a change in ionic strength,
or the like. In a particular embodiment, the dissociating agent is
a chemical dissociating agent that interacts with a portion of an
immobilized complex, thereby causing a portion of the immobilized
complex to dissociate. One specific example of a chemical
dissociating agent useful for practicing certain embodiments of the
invention is beta-mercaptoethanol. Dissociating agents are further
discussed in more detail below. In some embodiments, as will be
discussed more below, the dissociated species may comprise a
detectable component or a reactive component (e.g., an enzymatic
component) that is able to interact with a precursor labeling agent
molecule (e.g., an enzymatic substrate) to produce a labeling
agent.
[0069] In some embodiments, the immobilized complex comprises a
cleavable linkage. A "cleavable linkage," as used herein, is
linkage that is able to be readily (i.e. under conditions not
detrimental to the integrity of other portions of the immobilized
complex) and selectively cleaved upon exposure to a dissociating
agent. The cleavable linkage upon cleavage by exposure to a
dissociating agent forms the dissociated species. One specific
example of a cleavable linkage, which can be cleaved using
beta-mercaptoethanol, is a disulfide linkage. Cleavable linkages
and corresponding dissociating agents that can cause the cleavable
linkage to cleave are discussed in more detail below.
[0070] A cleavable linkage may be present in any portion of an
immobilized complex. For example, FIG. 4, panel (A), depicts an
immobilized complex 90 wherein the capture component 92 comprises a
cleavable linkage 94. Cleaving the cleavable linkage forms the
dissociated species 96 comprising the binding ligand 64, the
analyte molecule 62 and a portion 97 of the capture component,
wherein the complementary portion 98 of the capture component
remains attached to the substrate. FIG. 4, panel (B), depicts an
immobilized complex 100 wherein analyte molecule 102 comprises a
cleavable linkage. Cleaving of the cleavable linkage forms the
dissociated species 106 comprising the binding ligand 64 and a
portion 105 of the analyte molecule, wherein the complementary
portion 107 of the analyte molecule and the capture component 60
remain form a portion 108, which remains immobilized with respect
to the substrate. FIG. 4, panel (C), depicts immobilized complex
110 wherein binding ligand 112 comprises cleavable linkage 114.
Cleaving the cleavable linkage forms the dissociated species 116
comprising a portion of the binding ligand wherein the
complementary portion 117 of the binding ligand, the analyte
molecule 62, and the capture component 60 form a portion 118, which
remains immobilized with respect to the substrate.
[0071] In certain embodiments, at least some of the plurality of
dissociated species released from a substrate are partitioned
across a plurality of reaction sites, such as, for example, a
plurality of reaction vessels (e.g., in an array format). The
plurality of reaction vessels may be formed in or of any suitable
material, and in some cases, the reaction vessels can be sealed or
may be formed upon the mating of a substrate with a sealing
component, as discussed in more detail below. In certain
embodiments, especially where quantization of the analyte molecules
is desired, the partitioning of the dissociated species is
performed such that at least some (e.g., a statistically
significant fraction) of the reaction vessels comprise at least one
or, in certain cases, one dissociated species and at least some
(e.g., a statistically significant fraction) of the reaction vessel
comprise no dissociated species. The detectable species may be
quantified in certain embodiments, thereby allowing for the
detection and quantification of the analyte molecule in the fluid
sample by techniques described in more detail below. In certain
embodiments, the plurality of reactions vessels may comprise
detection capture components, wherein the detection capture
components are molecules or other entities able to specifically
bind at least one dissociated species. The plurality of dissociated
species may be detected in the reaction vessels, either indirectly
or directly, as discussed more below.
[0072] Certain methods of the present invention may be useful for
characterizing analyte molecules in a sample. In some cases, the
methods may be useful for detecting and/or quantifying analyte
molecules in a fluid sample which is suspected of containing at
least one analyte molecule, since, as explained in more detail
below, the number of reaction vessels which contain one or more of
the dissociated species can be correlated to the concentration of
analyte molecules in the fluid sample (e.g., the number of reaction
vessels which comprise a dissociated species can be related to a
measure of the concentration of the analyte molecules in the
sample). Certain embodiments of present invention thus can provide
a measure of the concentration of analyte molecules in a fluid
sample based on the proportion of reaction vessels which contain a
dissociated species. Specific methods and calculations of how to
quantify analyte molecules in a fluid sample using embodiments of
the invention are discussed more below.
Exemplary Basic Assay Formats
[0073] The inventive assays may be carried our according to a very
wide variety of basic protocols and formats. The particular format
chosen can be based on the nature of the analyte molecules and the
availability and properties of binding partners of the analyte as
well as other factors. Several exemplary basic formats were
discussed previously in the context of the discussion of FIGS. 1-4.
As would be apparent to those skilled in the art with the benefit
of the teachings provided by the present disclosure, the invention
may alternatively be performed according to protocols/formats not
specifically described in the specific, exemplary embodiments
illustrated in this detailed description, but which do not require
undue burden or experimentation to practice.
[0074] In certain embodiments, a method for determining a measure
of the concentration of analyte molecules or particles in a fluid
sample is provided that comprises capturing a plurality of analyte
molecules or particles (e.g., though association with a capture
component) on a first substrate (e.g., beads, microtiter plate,
etc.). A plurality of molecules or particles is released (e.g.,
dissociated) from the first substrate. At least a portion of the
molecules or particles released (dissociated species) from the
first substrate are detected on or within a second substrate, which
in certain embodiments comprises a plurality of reaction vessels
(e.g., an etched end of a fiber optic bundle--see below). A measure
of the concentration of the analyte molecules or particles in the
initial fluid sample can then be determined based on a measure of
quantification of the molecules or particles on or within the
second substrate that were released from the first substrate.
[0075] In certain embodiments, a method for determining a measure
of the concentration of analyte molecules or particles in a fluid
sample comprises first capturing a plurality of analyte molecules
or particles on a first substrate then releasing a plurality of
molecules or particles (disassociated species) from the first
substrate (e.g., immobilizing analyte molecules or particles with
respect to a capture component on a first substrate to form a
plurality of complexes and then releasing at least a portion of
some of the complexes formed). In certain embodiments, at least one
binding ligand comprising a binding partner for the molecule or
particle released from the first substrate is immobilized with
respect to each of at least a fraction of the plurality of
molecules or particles released from the first substrate to form a
plurality of complexes. In certain such cases, at least one of the
binding ligands may advantageously comprise a particle to
facilitate or enhance the performance of the assay, for example by
one or more of: increasing the degree of capture of and/or
enhancing the binding kinetics with the dissociated species;
facilitating transfer of the disassociated species from the first
substrate to the second substrate; and/or facilitating detection of
the disassociated species on or within the second substrate. The
plurality of complexes released from the first substrate can then
be detected and quantified (e.g., by partitioning the plurality of
dissociated species comprising particles across the plurality of
reaction vessels), and a measure of the concentration of the
analyte molecules or particles in the fluid sample may be
determined based on the quantification of the dissociated
species.
[0076] An example of the above embodiment is depicted in FIG. 5. A
plurality of analyte molecules or particles is captured on a first
substrate 230 to form a plurality of immobilized complexes 232
(step (A)). At least a portion of some of the immobilized complexes
are dissociated from the first substrate 230 and form a plurality
of dissociated species 236 (step (B)). In some cases, the plurality
of dissociated species may be formed by exposing the immobilized
complexes to a reducing agent and/or by causing a cleavable linker
located in the immobilized complex to cleave. In some cases, a
portion of the immobilized complexes 234 may remain associated with
the first substrate 230. The plurality of dissociated species may
be detected and/or quantified, in some cases, by partitioning at
least a portion of the plurality of dissociated species across a
plurality of reaction vessels (step (C)). A measure of the
concentration of the analyte molecules or particles in the fluid
sample may be determined based on the quantification of dissociated
species.
[0077] In some embodiments, the plurality of dissociated species
may be exposed to a plurality of binding ligands. At least one
binding ligand 240 comprising a binding partner for the molecule or
particle released from the first substrate may be immobilized with
respect to at least a fraction of the plurality of dissociated
species 236 from the first substrate to form a plurality of
complexes 244 forming a two-component dissociated species (step
(D)). An excess of binding ligands 246 may be present in some
cases. The plurality of two-component dissociated species 244 may
then be detected and/or quantified, in some cases, by partitioning
at least a portion of the plurality of complexes across a plurality
of reaction vessels 242 (step (E)). In some cases, the plurality of
binding ligands 246 that do not associate with a dissociated
species may also be partitioned across the plurality of reaction
vessels 242. A measure of the concentration of the analyte
molecules or particles in the fluid sample may be determined based
on the quantification of the complexes comprising a binding ligand
and a dissociated species.
[0078] In certain embodiments, a method for determining a measure
of the concentration of analyte molecules or particles in a fluid
sample may be performed as described in any of the methods above
with at least a fraction of the molecules or particles released
from the first substrate (disassociated species) becoming
immobilized with respect to the second substrate. For example, the
second substrate may comprise a plurality of capture components
with which the dissociated species may become associated. For
example, as depicted in FIG. 6, second substrate 250 comprising a
plurality of capture components 252 may be exposed to a plurality
of dissociated species 254 (step (A)). At least a portion of
dissociated species 254 may associate with capture components 252
on second substrate 250 to form complexes 256 (step (B)).
[0079] In some embodiments, the plurality of molecules may be
released from the first substrate by exposure to a dissociating
agent. For example, a substrate comprising a plurality of capture
components may be exposed to a sample comprising a plurality of
analyte molecules or particles, such that analyte molecules or
particles associate with capture components to form a plurality of
complexes, which are immobilized with respect to the substrate.
Each of the immobilized complexes may comprise at least one capture
component and at least one analyte molecule or particle. Exposure
of the plurality of immobilized complexes to a reducing agent
(e.g., beta-mercaptoethanol, dithiothreitol,
tris(2-carboxyethyl)phosphine, etc.) causes at least a portion of
at least some of the plurality of immobilized complexes to
dissociate from the substrate to form a plurality of dissociated
species. At least some of the dissociated species may be detected
to determine the presence of and/or a measurement of the amount or
concentration of the analyte molecules or particles in the fluid
sample, as discussed more herein. The reducing agent may or may not
be removed form the solution comprising the dissociated species
prior to detection of the dissociated species, as discussed more
herein. In some embodiments, the dissociating agent is a reducing
agent (e.g., beta-mercaptoethanol). In some embodiments, the
dissociating agent has essentially no specific affinity for the
capture components. That is, the dissociating agent does not bring
about release of the dissociating species by interacting with the
capture component and employing competitive binding to release the
analyte molecule that associated with the capture component.
[0080] The above example is illustrated in FIG. 7. First substrate
215 comprising a plurality of capture components 216 is exposed to
a plurality of analyte molecules 217 (step (A)). At least some of
the analyte molecules 217 associate with a capture component 216 on
first substrate 215 to form a plurality of immobilized complexes
218 (step (B)). The first substrate is then exposed to a
dissociating agent (e.g., a reducing agent) 219 and at least a
portion of the immobilized complexes are dissociated to form a
plurality of dissociated species 220 (step (C)). The complementary
portion 221 of the immobilized complexes may remain associated with
first substrate 215. The plurality of dissociated species 220 are
then exposed to second substrate 222 comprising a plurality of
capture components 223 which are able to specifically bind the
dissociated species (step (D)). At least some the dissociated
species 220 are immobilized with respect to a capture component 223
(step (E)) and at least some of the dissociated species may be
detected to determine the presence of and/or a measurement of the
amount or concentration of the analyte molecules or particles in
the fluid sample.
[0081] In some embodiments, the plurality of dissociated species
may be formed by cleavage of cleavable linkages. For example, each
of the immobilized complexes may comprise at least one cleavable
linkage (e.g., a disulfide linkage). The cleavable linkage may
located in a capture component, analyte molecule or a binding
ligand and may be cleaved to form a plurality of dissociated
species, for example, see FIG. 4 as discussed more herein. In a
particular embodiment, the cleavable linkage is a disulfide linkage
which may, in some cases, be cleaved by exposure of the immobilized
complexes to a reducing agent.
[0082] In some embodiments, at least a portion of an immobilized
complex comprises an enzymatic component. That is, at least one of
the capture component, the analyte molecule or any additional
components of the immobilized complex (e.g., binding ligand(s))
comprises an enzymatic component. In some cases, the enzymatic
component may be located in the portion of the immobilized complex
which is dissociated from the first substrate to form a dissociated
species. For example, FIG. 8 illustrate an exemplary embodiment of
an assay wherein the binding ligand comprises a moiety (e.g., an
enzymatic component), as discussed more herein.
[0083] In certain embodiments, the protocol may include the use of
at least one binding ligand, at least a portion of which comprises
at least a portion of the dissociated species transferred from the
first substrate to the second substrate (e.g., the binding ligand
may be immobilized prior to release or following release of the
molecules or particles from the first substrate). In some
embodiments, the binding ligand comprises a cleavable linkage
(e.g., a disulfide linkage) and/or is dissociated from the first
substrate by exposure to a reducing agent. In some embodiments, at
least one binding ligand comprises an enzymatic component. For
example, the binding ligand(s), or at least the portions thereof
forming at least a portion of the dissociated species transferred
from the first substrate to the second substrate, may further
comprise a moiety (e.g., an enzymatic component or enzyme
substrate) able to convert a precursor labeling agent molecule
(e.g., an enzymatic substrate) into a labeling agent (e.g., a
detectable product). After transfer of and, optionally, capture of
the dissociated species on or within the second substrate, the
second substrate may be exposed to a plurality of precursor
labeling agent molecules, wherein the plurality of precursor
labeling agent molecules are converted to a plurality of labeling
agent molecules upon exposure to a binding ligand. A measure of the
concentration of the analyte molecules or particles in the fluid
sample can then be determined based on the measurement of the
labeling agent molecules on or within the second substrate.
[0084] For example, as shown in FIG. 8, a plurality of analyte
molecules are immobilized with respect to first substrate 262 to
form a plurality of immobilized complexes 260 (step (A)). First
substrate 262 is exposed to a plurality of binding ligands 264
which associate with immobilized complexes 260 (step (B)). In this
illustrated example, a portion of at least some of binding ligands
264 are dissociated from the first substrate to form a plurality of
dissociated species 268 (step (C)) (e.g., by exposure to a
dissociating agent and/or by cleavage of a cleavable linker). Each
dissociated species 268 comprises a moiety 266 which is able to
convert a precursor labeling agent molecule into a labeling agent.
The plurality of dissociated species 266 is immobilized with
respect to second substrate 270 (step (D)). The second substrate is
exposed to a plurality of precursor labeling agent molecules 272,
at least some of which are converted to a labeling agent molecules
274 upon exposure to moiety 268 of dissociated species 266, as
indicated by arrow 276 (step (E)).
[0085] As another example, as shown in FIG. 9, a plurality of
analyte molecules are immobilized with respect to first substrate
282 to form a plurality of immobilized complexes 280 (step (A)). At
least a portion of at least some of the immobilized complexes are
dissociated from first substrate 282 to form a plurality of
dissociated species 284 (step (B)) (e.g., by exposure to a
dissociating agent and/or by cleavage of a cleavable linker). A
plurality of dissociated species 284 may be exposed to a plurality
of binding ligands 286 (step (C)). Binding ligands 286 may
associated with at least some of the dissociated species 284 to
form a plurality of complexes 288 forming a two-component
dissociated species (i.e. binding ligand 286 and species 284) (step
(D)). In some cases, binding ligand 286 may comprise a moiety 290
that is able to convert a precursor labeling agent molecule into a
labeling agent. The detection and/or quantification of the
dissociated species (comprising a binding ligand) may be carried
out according to the methods discussed herein.
[0086] In certain method of the invention substantially all of the
detected dissociated species that are applied to the second
substrate become spatially separated with respect to the other
detected dissociated species during detection (e.g., by being
located in separate and distinct areas of the second substrate,
such as separate reaction vessels), so that detection of the
dissociated species is able to resolve individual dissociated
species (i.e. individual molecules or particles) of the plurality
of dissociated species. The ability to achieve spatially separated
species may aid in the determination of a measure of the
concentration of analyte molecules in a fluid sample and/or the
detection of a plurality of species substantially simultaneously.
The ability to spatially separate a plurality of dissociated
species may permit low numbers of dissociated species to be
detected and quantified, thereby enabling measurement of very low
concentrations of analyte molecules in a fluid sample to be
tested.
[0087] In some embodiments, a system may be provided comprising a
first substrate comprising a plurality of capture components and a
second substrate configured to receive and contain dissociated
species therein or thereon. The system can additionally comprise a
detector configured to detect dissociated species in or on the
second substrate. The plurality of capture components may be able
to become specifically immobilized with respect to a plurality of
analyte molecules or particles in the fluid sample, such that the
analyte molecules or particles associate with capture components to
form a plurality of immobilized complexes. At least a portion of at
least some of the complexes is susceptible to being dissociated
from the first substrate by exposure to a reducing agent, to form a
plurality of dissociated species.
[0088] In certain embodiments, the systems and/or methods of the
present invention can be employed using precursor labeling agent
molecules, wherein the precursor labeling agent molecules are
converted to labeling agent molecules which are insoluble in the
liquid substantially surrounding the labeling agent molecules
(e.g., the liquid in the reaction vessel) and/or which become
immobilized with respect to the second substrate (e.g., within the
reaction vessel). For example, the systems and/or methods of the
present invention may be used in combination with the systems
and/or methods as described in commonly owned U.S. patent
application Ser. No. (not yet determined), filed Sep. 23, 2008,
entitled "High Sensitivity Determination of the Concentration of
Analyte Molecules or Particles in a Fluid Sample" by Duffy, et al.
(Attorney Docket No. Q0052.70006US00).
Systems and Kits
[0089] In some embodiments of the present invention, a system for
detecting analyte molecules or particles in a fluid sample is
provided. The system may comprise a first substrate comprising a
plurality of capture components configured for specifically
immobilizing the analyte molecules or particles with respect to the
substrate to form a plurality of immobilized complexes. The
complexes may be configured to be susceptible to being dissociated
from the first substrate by a dissociating agent, e.g., by cleaving
of a cleavable linker to form a plurality of dissociated species.
The system may further comprise a second substrate comprising an
array comprising a plurality of reaction vessels. The reaction
vessels may be configured to receive and contain dissociated
species therein.
[0090] In certain embodiments, a system for detecting analyte
molecules or particles in a fluid sample may comprise a first
substrate comprising a plurality of capture components able to
become specifically immobilized with respect to a plurality of
analyte molecules or particles in the fluid sample, such that the
analyte molecules or particles associate with capture components to
form a plurality of immobilized complexes. At least a portion of
the some of the immobilized complexes, in this embodiment, may be
susceptible to being dissociated from the first substrate by
exposure to a reducing agent, to form a plurality of dissociated
species. The system may also comprise a second substrate configured
to receive and contain dissociated species therein or thereon and a
detector configured to detect dissociated species in or on the
second substrate.
[0091] In some embodiments, the first substrate may comprise a
plurality of beads 900 (e.g., magnetic beads), a planar surface
902, or a microtiter plate 904, as shown in FIGS. 10A-10C,
respectively, and as discussed in more detail herein. Second
substrates may comprise a plurality of reaction vessels are also
discussed in more detail herein. In a particular embodiment, the
plurality of reaction vessels may be formed in the end of a fiber
optic bundle. The volume of each of the reactions vessels of the
second substrate may be between about 10 attoliters and about 100
picoliters or between about 10 femtoliters and about 100
femtoliters, in certain embodiments, as discussed previously. In
some embodiments, the plurality of reaction vessels may comprise a
plurality of dissociated species capture components to which
dissociated species are able to become specifically immobilized.
The second substrate may, in some embodiments, further comprise a
sealing component configured for sealing the plurality of reaction
vessels. In certain embodiments, the second substrate comprises a
substantially planar surface and the sealing component comprises a
plurality of microwells, such that a plurality of reaction vessels
is formed upon the mating of at least a portion of the planar
surface with at least a portion of the sealing component comprising
the plurality of microwells. In certain embodiments, the plurality
of reaction vessels may be formed upon the mating of at least a
portion of a sealing component and at least a portion of the second
substrate, as shown in FIGS. 11A-11F and as discussed in more
detail herein.
Capture Components
[0092] In some embodiments of the present invention, the surfaces
of a substrate (e.g., the substrate on which the analyte molecules
or particles form an immobilized complex ("first substrate") or the
substrate to which the dissociated species are transferred ("second
substrate")) may, as mentioned previously, incorporate at least one
type of capture component. As mentioned above, a capture component
is any molecule, other chemical/biological entity or solid support
modification disposed upon a solid support that can be used to
specifically attach, bind or otherwise capture a target molecule or
particle (e.g., an analyte molecule or dissociated species), such
that the target molecule/particle becomes immobilized with respect
to the capture component and solid substrate. Generally, the
capture component allows the attachment of a molecule, particle or
complex to a solid support (that is, a surface of a substrate) for
the purposes of immobilization, detection, quantification, and/or
other analysis of the molecule, particle or complex. A capture
component is used in the present invention, in some cases, to
immobilize an analyte molecule with respect to the first substrate.
In some cases, a capture component may comprise a cleavable
linkage, as discussed more below. Although much of the following
discussion focuses on a capture component with respect to the
immobilization of an analyte molecule, it should be understood that
the same types of capture components may, as appropriate, be used
to immobilize dissociated species as well. Those of ordinary skill
in the art will be able to select appropriate capture component in
accordance with the material (e.g., analyte molecule or dissociated
species) to be immobilized.
[0093] As will be appreciated by those in the art, the composition
of the capture component will depend on the composition of the
analyte molecule (or dissociated species). Capture components for a
wide variety of target molecules are known or can be readily found
using known techniques. For example, when the target molecule is a
protein, the capture components may comprise proteins, particularly
antibodies or fragments thereof (e.g., antigen-binding fragments
(Fabs), Fab' fragments, pepsin fragments, F(ab').sub.2 fragments,
full-length polyclonal or monoclonal antibodies, antibody-like
fragments, etc.), other proteins, such as receptor proteins,
Protein A, Protein C, etc., or small molecules. In some cases,
capture components for proteins comprise peptides. For example,
when the target molecule is an enzyme, suitable capture components
may include enzyme substrates and enzyme inhibitors. In some cases,
when the target analyte is a phosphorylated species, the capture
component may comprise a phosphate-binding agent. For example, the
phosphate-binding agent may comprise metal-ion affinity media such
as those describe in U.S. Pat. No. 7,070,921 and U.S. Patent
Application No. 20060121544. In addition, when the target molecule
is a single-stranded nucleic acid, the capture component may be a
complementary nucleic acid. Similarly, the target molecule may be a
nucleic acid binding protein and the capture component may be a
single-stranded or double-stranded nucleic acid; alternatively, the
capture component may be a nucleic acid-binding protein when the
target molecule is a single or double stranded nucleic acid.
Alternatively, as is generally described in U.S. Pat. Nos.
5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867,
5,705,337, and related patents, nucleic acid "aptamers" may be
developed for capturing virtually any target molecule. As will be
appreciated by those or ordinary skill in the art, any molecule
that can specifically associate with a target molecule of interest
may potentially be used as a capture component. For example, when
the target molecule is a carbohydrate, potentially suitable capture
components include, for example, antibodies, lectins and
selectins.
[0094] For certain embodiments, suitable target molecule (e.g.,
analyte molecule or dissociated species)/capture component pairs
can include, but are not limited to, antibodies/antigens,
receptors/ligands, proteins/nucleic acid, nucleic acids/nucleic
acids, enzymes/substrates and/or inhibitors, carbohydrates
(including glycoproteins and glycolipids)/lectins and/or selectins,
proteins/proteins, proteins/small molecules; small molecules/small
molecules, etc. According to one embodiment, the capture components
are portions (particularly the extracellular portions) of cell
surface receptors that are known to multimerize, such as the growth
hormone receptor, glucose transporters (particularly GLUT 4
receptor), and T-cell receptors and the target analytes are one or
more receptor target ligands.
[0095] In a particular embodiment, the capture component may be
attached to a binding surface (for example, the surface of a
microwell or of a microbead) via a linkage, which may comprise any
moiety, functionalization, or modification of the binding surface
and/or capture component that facilitates the attachment of the
capture component to the surface. The linkage between the capture
component and the surface may comprise one or more chemical or
physical (e.g., non-specific attachment via van der Waals forces,
hydrogen bonding, electrostatic interactions,
hydrophobic/hydrophilic interactions; etc.) bonds and/or chemical
linkers providing such bond(s). In certain embodiments, the capture
component comprises a capture extender component. In such
embodiments, the capture component comprises a first portion that
binds the analyte molecule and a second portion that can be used
for attachment to the binding surface.
[0096] In certain embodiments, the substrate surface may also
comprise a protective or passivating layer that can reduce or
minimize non-specific attachment of non-capture components (e.g.,
analyte molecules, binding ligands, dissociated species) to the
binding surface during the assay which may lead to false positive
signals during detection or to loss of signal. Examples of
materials that may be utilized in certain embodiments to form
passivating layers include, but are not limited to: polymers, such
as poly(ethylene glycol), that repel the non-specific binding of
proteins; naturally occurring proteins with this property, such as
serum albumin and casein; surfactants, e.g. zwitterionic
surfactants, such as sulfobetaines; naturally occurring long-chain
lipids; and nucleic acids, such as salmon sperm DNA.
[0097] The method of attachment of the capture component to the
substrate surface depends of the type of linkage employed and may
potentially be accomplished by a wide variety of suitable coupling
chemistries/techniques known to those of ordinary skill in the art.
The particular means of attachment selected will depend on the
material characteristics of the substrate surface and the nature of
the capture component. In certain embodiments, the capture
components may be attached to the substrate surface through the use
of reactive functional groups on each. According to one embodiment,
the functional groups are chemical functionalities. That is, the
binding surface may be derivatized such that a chemical
functionality is presented at the binding surface which can react
with a chemical functionality on the capture component resulting in
attachment. Examples of functional groups for attachment that may
be useful include, but are not limited to, amino groups, carboxy
groups, epoxide groups, maleimide groups, oxo groups and thiol
groups. Functional groups can be attached, either directly or
through the use of a linker, the combination of which is sometimes
referred to herein as a "crosslinker." Crosslinkers are known in
the art; for example, homo-or hetero-bifunctional crosslinkers as
are well known (e.g., see 1994 Pierce Chemical. Company catalog,
technical section on crosslinkers, pages 155-200, or "Bioconjugate
Techniques" by Greg T. Hermanson, Academic Press, 1996).
Non-limiting example of crosslinkers include alkyl groups
(including substituted alkyl groups and alkyl groups containing
heteroatom moieties), esters, amide, amine, epoxy groups and
ethylene glycol and derivatives. A linker may also be a sulfone
group, forming a sulfonamide.
[0098] According to one embodiment, the functional group is a
light-activated functional group. That is, the functional group can
be activated by light to attach the capture component to the
substrate surface. One example is PhotoLink.TM. technology
available from SurModics, Inc. in Eden Prairie, Minn.
[0099] In some cases, the substrate may comprise
streptavidin-coated surfaces and the capture component may be
biotinylated. Exposure of the capture component to the
streptavidin-coated surfaces can cause association of the capture
component with the surface by interaction between the biotin
component and streptavidin.
[0100] In certain embodiments, attachment of the capture component
to the binding surface may be effected without covalently modifying
the binding surface of a substrate. For example, the attachment
functionality can be added to the binding surface by using a linker
that has both a functional group reactive with the capture
component and a group that has binding affinity for the binding
surface. In certain embodiments, a linker comprises a protein
capable of binding or sticking to the binding surface; for example,
in one such embodiment, the linker is serum albumin with free amine
groups on its surface. A second linker (crosslinker) can then be
added to attach the amine groups of the albumin to the capture
component (e.g., to carboxy groups).
[0101] According to one embodiment in which a chemical crosslinker
is used to attach the capture components to the substrate, the
analyte molecule may be captured on the binding surface of a
substrate using a capture component attached via chemical
crosslinking in the following manner. First, the binding surface is
derivatized with a functional group, such as, an amine group. Next,
a crosslinker and the capture component are placed in contact with
the binding surface such that one end of the crosslinker attaches
to the amine group and the capture component attaches to the other
end of the crosslinker. In this way, capture components comprising
proteins, lectins, nucleic acids, small organic molecules,
carbohydrates can be attached.
[0102] One embodiment utilizes proteinaceous capture components. As
is known in the art, any number of techniques may be used to attach
a proteinaceous capture component to a wide variety of solid
surfaces. "Protein" or "proteinaceous" in this context includes
proteins, polypeptides, peptides, including, for example, enzymes
and antibodies. A wide variety of techniques are known to add
reactive moieties to proteins, for example, the method outlined in
U.S. Pat. No. 5,620,850. The attachment of proteins to surfaces is
known, for example, see Heller, Acc. Chem. Res. 23:128 (1990), and
many other similar references.
[0103] In some embodiments, the capture component (or binding
ligand) may comprise Fab' fragments. The use of Fab' fragments as
opposed to whole antibodies may help reduce non-specific binding
between the capture component and the binding ligand. In some
cases, the Fc region of a capture component (or binding ligand) may
be removed (e.g., proteolytically). In some cases, an enzyme may be
used to remove the Fc region (e.g., pepsin, which may produce
F(ab').sub.2 fragments and papain, which may produce Fab
fragments). In some instances, the capture component may be
attached to a binding surface using amines or may be modified with
biotin (e.g., using NHS-biotin) to facilitate binding to an avidin
or streptavidin coated substrate surface. F(ab').sub.2 fragments
may be subjected to a chemical reduction treatment (e.g., by
exposure to 2-mercaptoethylamine) to, in some cases, form two
thiol-bearing Fab' fragments. These thiol-bearing fragments can
then be attached via reaction with a Michael acceptor such as
maleimide. For example, the Fab' fragments may then be treated with
a reagent (e.g., maleimide-biotin) to attach at least one biotin
moiety (i.e., biotinylated) to facilitate attachment to
streptavidin-coated surfaces as described above.
[0104] Certain embodiments utilize nucleic acids as the capture
component, for example for when the analyte molecule is a nucleic
acid or a nucleic acid binding protein, or when the it is desired
that the capture component serve as an aptamer for binding a
protein, as is well known in the art.
[0105] According to one embodiment, each binding surface of a
substrate comprises a plurality of capture components. The
plurality of capture components, in some cases, may be distributed
randomly on the binding surface like a "lawn." Alternatively, the
capture components may be spatially segregated into distinct
region(s) and distributed in any desired fashion.
[0106] Binding between the capture component and the analyte
molecule, in certain embodiments, is specific, e.g., as when the
capture component and the analyte molecule are complementary parts
of a binding pair. In certain such embodiments, the capture
component binds both specifically and directly to the analyte
molecule. By "specifically bind" or "binding specificity," it is
meant that the capture component binds the analyte molecule with
specificity sufficient to differentiate between the analyte
molecule and other components or contaminants of the test sample.
For example, the capture component, according to one embodiment,
may be an antibody that binds specifically to some portion of an
analyte molecule (e.g., an antigen). The antibody, according to one
embodiment, can be any antibody capable of binding specifically to
an analyte molecule of interest. For example, appropriate
antibodies include, but are not limited to, monoclonal antibodies,
bispecific antibodies, minibodies, domain antibodies, synthetic
antibodies (sometimes referred to as antibody mimetics), chimeric
antibodies, humanized antibodies, antibody fusions (sometimes
referred to as "antibody conjugates"), and fragments of each,
respectively. As another example, the analyte molecule may be an
antibody and the capture component may be an antigen.
[0107] According to one embodiment in which an analyte particle is
a biological cell (e.g., mammalian, avian, reptilian, other
vertebrate, insect, yeast, bacterial, cell, etc.), the capture
component may be a ligand having specific affinity for a cell
surface antigen (e.g., a cell surface receptor). In one embodiment,
the capture component is an adhesion molecule receptor or portion
thereof, which has binding specificity for a cell adhesion molecule
expressed on the surface of a target cell type. In use, the
adhesion molecule receptor binds with an adhesion molecule on the
extracellular surface of the target cell, thereby immobilizing or
capturing the cell. In one embodiment in which the analyte particle
is a cell, the capture component is fibronectin, which has
specificity for, for example, analyte particles comprising neural
cells.
[0108] In some embodiments, as will be appreciated by those of
ordinary skill in the art, it is possible to detect analyte
molecules using capture components for which binding to analyte
molecules/dissociated species that is not highly specific. For
example, such systems/methods may use different capture components
such as, for example, a panel of different binding ligands, and
detection of any particular analyte molecule/dissociated species is
determined via a "signature" of binding to this panel of binding
ligands, similar to the manner in which "electronic noses" work.
This may find particular utility in the detection of certain small
molecule analytes. In some embodiments, the binding affinity
between analyte molecules and capture components should be
sufficient to remain bound under the conditions of the assay,
including wash steps to remove molecules or particles that are
non-specifically bound. In some cases, for example in the detection
of certain biomolecules, the binding constant of the analyte
molecule or dissociated species to its complementary capture
component may be between at least about 10.sup.4 and about 10.sup.6
M.sup.-1, at least about 10.sup.5 and about 10.sup.9 M.sup.-1, at
least about 10.sup.7 and about 10.sup.9 M.sup.-1, greater than
about 10.sup.9 M.sup.-1, or the like.
[0109] In certain embodiments, the capture component is chosen to
be able to bind to a corresponding binding partner associated with
or attached to the analyte molecule or dissociated species. For
example, the capture component according to one embodiment is a
chemical crosslinker as described above able to bind to proteins
generally. According to one embodiment, every protein molecule in a
fluid sample comprises an analyte molecule that attaches to such a
chemical crosslinker. In another example, the capture component
comprises streptavidin, which binds with high affinity to biotin,
and thus captures any analyte molecules to which biotin has been
attached. Alternatively, the capture component may be biotin, and
streptavidin may be attached to or associated with the analyte
molecules such that the analyte molecules can be captured by the
biotin.
[0110] According to one embodiment, the binding surfaces of a
substrate may be functionalized with capture components in the
following manner. First, the surface of a substrate (e.g., one end
of a fiber optic bundle, a plurality of microbeads, etc.) is
prepared for attachment of the capture component(s) by being
modified to form or directly bind to the capture components, or a
linker may be added to the binding surface of the substrate such
that the capture component(s) attaches to the binding surface of
the substrate via the linker. In one embodiment, the binding
surfaces of the substrate are derivatized with a chemical
functionality as described above. Next, the capture component may
be added, which binds to and is immobilized by the chemical
functionality.
[0111] A specific embodiment is depicted in FIG. 12, in which the
binding surface comprises an array of microwells functionalized
with biotin. As shown in panel (A), an array of microwells 130 in
this non-limiting example is formed at the end of a fiber optic
bundle 126. To attach the capture component, the binding surface of
the microwells 130 are first modified (e.g., with aminopropyl
silane), as indicated by arrow 127, which may be bound to both the
core 131 and cladding 132 surfaces of the end of the fiber bundle
126, as shown in FIG. 12, panel (B). However, in certain
embodiments, the capture component should be present only within
the microwells, the external surfaces of the fiber optic bundle,
such as the external surfaces 133 of the cladding 132, should not
be modified. In certain cases, after treatment, chemical
functionalities may be removed from the external cladding surface
133 to avoid attachment of a capture component in this region. In
this example, as shown in FIG. 12, panel (C), and as indicated by
arrow 129, treated binding surface 128 may be removed from the
external cladding portion 133, e.g., by polishing the tip of the
fiber optical bundle (e.g., for 10 seconds with 0.3 um lapping
film), thereby removing the topmost layer of the cladding in this
region, thereby removing the added binding moieties. After
functionalization of the binding surface of the microwells, the
capture component can be attached, as indicated by arrow 131 and
shown in FIG. 12, panel (D). In one embodiment, the surface is
treated with aminopropyl silane and the capture component comprises
biotin or is labeled with biotin. For example, referring to FIG.
12, panel (D), a capture component comprising biotin succinimidyl
ester 136 is attached to the amino groups of treated surface 128 of
the microwells 130. The modification with aminopropyl silane is
effective in this example because NHS-biotin attaches to an
amino-silanized binding surface 128.
[0112] Examples of capture components 146 and exemplary association
of capture components with an analyte molecule 142 within a
reaction vessel 130 are depicted in FIG. 12, panels (E-G). A
capture component 146 may be localized directly on the surface of
the microwell 130, which may contain an optional seal 138, on a
microparticle 134 contained within the microwell 130 (FIG. 12,
panel (F)), and/or on the seal 138 of the microwell 130 (FIG. 12,
panel (G)). Additional locations where a capture component may be
immobilized and additional substrates that may be used for one or
both of capturing an analyte molecule or a dissociated species
according to certain methods of the invention are discussed more
below.
[0113] In some embodiments only a single analyte molecule
associates with each capture component. However, in some instances,
more than one analyte molecule may be immobilized with respect to
each capture component, in which case, an immobilized complex may
comprise a capture component and at least two analyte molecules. In
yet other cases, a single analyte molecule may become immobilized
with respect to two or more capture components (either of the same
or differing types) such that an immobilized complex comprises at
least two capture component and an analyte molecule. In some cases,
at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, at least about 95%, at least
about 97%, at least about 99%, or the like, of analyte molecules in
the fluid sample exposed to the binding substrate become
immobilized with respect to at least one capture component.
Substrates
[0114] In some embodiments, the method and systems of the present
invention comprise a first substrate and a second substrate. In
certain embodiments, the first substrate comprises a plurality of
capture component with specificity for target analyte molecules (or
particles) in a fluid sample to be tested, while the second
substrate comprises a plurality of capture component with
specificity for dissociated species released from the first
substrate. The second substrate in certain embodiments comprises
the substrate utilized for detection and quantization and to
facilitate this comprises a plurality of reaction vessels in which
a plurality of dissociated species (or molecules or particles)
released from first substrate are partitioned across or received
and contained in. The discussion below will first focus on the
first substrate, and then focus on the second substrate. It should
be understood, that the first substrate and the second substrate,
in some embodiments, may be interchanged with each other, may be
essentially the same in configuration or may be different in
configuration.
[0115] The first substrate may be fabricated from any suitable
material, for example, plastics, polysaccharides, nylon or
nitrocellulose, composite materials, ceramics, silica or
silica-based materials, carbon, metals, inorganic glasses, and a
variety of other polymers. Non-limiting examples of potentially
suitable configurations include of beads (e.g., magnetic beads),
microtiter plates, slides, membranes, plates, flasks, dipsticks,
optical fibers, or the like. In some embodiments, the first
substrate may comprise a planar surface 902, for example, as shown
in FIG. 10B.
[0116] In a particular embodiment, the first substrate comprises a
plurality of beads 900 (FIG. 10A). The beads may each comprise a
plurality of capture components by which a plurality of analyte
molecules may be immobilized. Without wishing to be bound by any
theory, the use of beads or other large surface area formats as the
first substrate may increase the speed and/or efficiency in which a
plurality of analyte molecules are immobilized with respect to the
substrate. This is because the solution comprising the plurality of
analyte molecules and beads may be agitated (e.g., stirred) and may
increase the rate of exposure of the substrate (e.g., bead) to an
analyte molecule, as compared to a first substrate comprising a
different configuration (e.g., a microtiter plate).
[0117] In some embodiments, the beads may be magnetic beads. The
magnetic property of the beads may help in separating the beads
from a solution (e.g., comprising a plurality of dissociated
species) and/or during washing step(s) (e.g., to remove excess
fluid sample, binding ligands, and/or dissociated species).
Potentially suitable beads, including magnetic beads, are available
from a number of commercial suppliers. In certain embodiments, the
diameter of the beads may be greater than about 0.1 um, greater
than about 1 um, greater than about 10 um, greater than about 100
um, greater than about 1 mm, or the like. In other embodiments, the
diameter of the beads may be between about 0.1 um and about 100 um,
between about 1 um and about 100 um, between about 10 um and about
100 um, between about 0.1 um and about 1 mm, between about 10 um
and about 10 mm, between about 0.1 um and about 10 um, or the
like.
[0118] In another set of embodiments, e.g. as illustrated in FIG.
10C, the first substrate comprises a microtiter plate 904, wherein
each well 906 of the plate comprises a plurality of capture
components. The volume of each well may be greater than about 1 nL,
greater than about 10 nL, greater than about 100 nL, greater than
about 1 uL, greater than about 10 uL, greater than about 100 uL,
greater than about 1 mL, greater than about 2 mL, or the like. In
other cases, the volume of each well may be between about 1 nL and
about 2 mL, between about 10 nL and about 1 mL, between about 100
nL and about 100 uL, between about 10 uL and about 1 mL, between
about 100 uL and about 1 mL, or the like. The number of wells in
the microtiter plate may be any appropriate number for example
about 6, about 24, about 96, about 384, about 1536, or the like.
The number of wells may be between about 5 and about 2000, about 10
and about 1000, about 20 and about 500, about 50 and about 100, or
the like. In some cases, the microtiter plate may be a 96-well
plate (FIG. 10C). Microtiter plates may be purchased from many
commercial sources.
[0119] In some embodiments, the second substrate is employed for
the detection and/or quantification of a plurality of dissociated
species (e.g., molecules or particles released from the first
substrate) and comprises a plurality of small volume reaction
vessels. The second substrate, in certain embodiments, is
configured to receive and contain dissociated species therein or
thereon (e.g., within wells comprising capture components having
specificity for the dissociated species, or within wells without
such capture components but including means for sealing of the
wells of the substrate--e.g., through use of a sealing component
(see below), etc.). The plurality of dissociated complexes can be
partitioned across a plurality of reaction vessels of the second
substrate (e.g., configured as an array of reaction vessels), in
some cases, to facilitate determination of a measure of the
concentration of dissociated species and thereby a measure of the
concentration of analyte molecules in a fluid sample by means
discussed in further detail below and in the examples.
[0120] In some embodiments of the present invention, the plurality
of reaction vessels may be formed through the mating of the second
substrate and a sealing component, wherein at least one of the
second substrate and the sealing component comprises a plurality of
microwells. In some cases, (e.g., as shown in FIG. 12, panels
(E-G)) an array comprises a plurality of depressions in a first
surface of the second substrate (e.g., a support material). The
sealing component may comprise a second surface with the same or
different topology as the first surface may be brought into contact
with the first surface to create a plurality of sealed reaction
vessels. Either the first surface or the second surface may be
fabricated from a compliant material to aid in sealing. Either or
both of the surfaces may be hydrophobic or contain hydrophobic
regions to minimize leakage of aqueous samples from the microwells.
In some cases, the sealing component may be capable of contacting
the exterior surface of an array of microwells (e.g., the cladding
of a fiber optic bundle as shown in FIG. 12) such that each
reaction vessel thus formed is sealed or isolated such that the
contents of each reaction vessel cannot escape the reaction vessel.
According to one embodiment, the sealing component may be a
silicone elastomer gasket that may be placed against an array of
microwells with application of uniform pressure across the entire
substrate. In some cases, the reaction vessels may be sealed after
the addition of a dissociated species and, optionally, a precursor
labeling agent molecule to facilitate detection of the dissociated
species. For embodiments employing precursor labeling agent
molecules, by sealing the contents in some of each reaction vessel,
a reaction to produce the detectable labeling agent molecule can
proceed within the sealed reaction vessels, thereby producing a
detectable amount of a labeling agent molecule that is retained in
the reaction vessel for detection purposes. In some cases, the
plurality of reaction vessels formed on a planar second substrate
upon the mating of at least a portion of a sealing component
comprising a plurality of microwells and at least a portion of the
planar substrate (e.g., see FIG. 11B below).
[0121] Non-limiting embodiments of the formation of a plurality of
reaction vessels on the second substrate are depicted in FIG. 11.
FIG. 11, panel (A) shows a surface comprising a plurality of
microwells 139, which have been exposed to a fluid sample 141, and
a sealing component 143. Sealing component 143 in this example
comprises a substantially planar bottom surface. Mating of the
microwell containing surface 139 with sealing component 143 forms a
plurality of sealed reaction vessels 145. The areas between the
reaction vessels 148, may be modified to aid in the formation of a
tight seal between the reaction vessels.
[0122] A second embodiment is shown in FIG. 11, panel (B), in which
sealing component 162 comprising a plurality of microwells 163 is
mated with a substantially planar surface 158 which has been
exposed to fluid sample 162, thereby forming a plurality of
reaction vessels 164.
[0123] In a third embodiment, as shown in FIG. 11, panel (C),
substrate surface 166 comprising a plurality of microwells 167 is
mated with sealing component 170 also comprising a plurality of
microwells 171. In this embodiment, the microwells in the substrate
and the microwells in the sealing components are substantially
aligned so each reaction vessel 172 formed comprises a portion of
the microwell from the sealing component and a portion of a
microwell from the substrate. In FIG. 11, panel (D), the microwells
are not aligned such that each reaction vessel comprises either a
microwell from the sealing component 173 or a microwell from the
substrate 175.
[0124] The sealing component may be essentially the same size as
the substrate or may be different in size. In some cases, the
sealing component is approximately the same size as the substrate
and mates with substantially the entire surface of the substrate.
In other cases, as depicted in FIG. 11, panel (E), the sealing
component 176 is smaller than the substrate 174 and the sealing
component only mates with a portion 178 of the substrate. In yet
another embodiment, as depicted in FIG. 11, panel (F), the sealing
component 182 is larger than the substrate 180, and only a portion
184 of the sealing component mates with the substrate 180.
[0125] In some embodiments, the reaction vessels may all have
approximately the same volume. In other embodiments, the reaction
vessels may be of differing volumes. The volume of each individual
reaction vessel can range for different embodiments from attoliters
or smaller to nanoliters or larger depending upon the nature of
dissociated species, the detection technique and equipment
employed, and the expected concentration of the dissociated species
in the fluid applied to the second substrate for analysis. In one
embodiment, the size of the reaction vessel may be selected such
that at the concentration of interest, between zero and ten
molecules/particles of a dissociated species would be statistically
expected to be found in each reaction vessel. In a particular
embodiment, the volume of the reaction vessel is selected such that
at the concentration of interest, either zero or one
molecules/particles of the dissociated species would be
statistically expected to be found in each reaction vessel. In
accordance with one embodiment of the present invention, the
reaction vessels may have a volume between about 1 femtoliter and
about 1 picoliter, between about 10 femtoliters and about 100
femtoliters, between about 10 attoliters and about 50 picoliters,
between about 1 picoliter and about 50 picoliters, between about 1
femtoliter and about 1 picoliter, between about 30 femtoliters and
about 60 femtoliters, or the like. In some cases, the reaction
vessels have a volume of less than about 1 picoliter, less than
about 500 femtoliters, less than about 100 femtoliters, less than
about 50 femtoliters, less than about 1 femtoliter, or the like. In
some cases, the reaction vessels have a volume of about 10
femtoliters, about 20 femtoliters, about 30 femtoliters, about 40
femtoliters, about 50 femtoliters, about 60 femtoliters, about 70
femtoliters, about 80 femtoliters, about 90 femtoliters, about 100
femtoliters, or the like.
[0126] The number of reaction vessels in the array will depend on
the composition and end use of the array. Arrays containing from
about 2 to many billions of reaction vessels can be made by
utilizing a variety of techniques and materials. Increasing the
number of reaction vessels in the array can be used to increase the
dynamic range of an assay or to allow multiple samples or multiple
types of dissociated species to be assayed in parallel. Generally,
the array will comprise between one thousand and one million
reaction vessels per sample to be analyzed. In some cases, the
array will comprise greater than one million reaction vessels. In
some embodiments, the array will comprise between about 1,000 and
about 50,000, between about 1,000 and about 1,000,000, between
about 1,000 and about 10,000, between about 10,000 and about
100,000, between about 100,000 and about 1,000,000, between about
1,000 and about 100,000, between about 50,000 and about 100,000,
between about 20,000 and about 80,000, between about 30,000 and
about 70,000, between about 40,000 and about 60,000, or about
50,000, or the like, reaction vessels.
[0127] The array of reaction vessels may be arranged on a
substantially planar surface or in a non-planar three-dimensional
arrangement. The reaction vessels may be arrayed in a regular
pattern or may be randomly distributed. In a specific embodiment,
the array is a regular pattern of sites on a substantially planar
surface permitting the sites to be addressed in the X-Y coordinate
plane.
[0128] In some embodiments, the reaction vessels are formed in a
solid material. As will be appreciated by those in the art, the
number of potentially suitable materials in which the reaction
vessels can be formed is very large, and includes, but is not
limited to, glass (including modified and/or functionalized glass),
plastics (including acrylics, polystyrene and copolymers of styrene
and other materials, polypropylene, polyethylene, polybutylene,
polyurethanes, Teflon.RTM., polysaccharides, nylon or
nitrocellulose, etc.), elastomers (such as poly(dimethyl siloxane)
and poly urethanes), composite materials, ceramics, silica or
silica-based materials (including silicon and modified silicon),
carbon, metals, optical fiber bundles, or the like. In general, the
substrate material may be selected to allow for optical detection
without appreciable autofluorescence. In certain embodiments, the
reaction vessels may be formed in a flexible material.
[0129] Individual reaction vessels may contain a binding surface.
The binding surface may comprise essentially the entirety or only a
portion of the interior surface of the reaction vessel or may be on
the surface of another material or object that is confined within
the reaction vessel, such as, for example, a bead, or a particle
(for example, a micro-particle or a nanoparticle).
[0130] A microwell in a surface (e.g., substrate or sealing
component) may be formed using a variety of techniques known in the
art, including, but not limited to, photolithography, stamping
techniques, molding techniques, etching techniques, or the like. As
will be appreciated by those of the ordinary skill in the art, the
technique used will depend on the composition and shape of the
supporting material and the size and number of reaction
vessels.
[0131] In a particular embodiment, an array of reaction vessels is
formed by creating microwells on one end of a fiber optic bundle
and utilizing a planar compliant surface as a sealing component. In
certain such embodiments, an array of reaction vessels in the end
of a fiber optic bundle may be formed as follows. First, an array
of microwells is etched into the end of a polished fiber optic
bundle. Techniques and materials for forming and etching a fiber
optic bundle are known to those of ordinary skill in the art. For
example, the diameter of the optical fibers, the presence, size and
composition of core and cladding regions of the fiber, and the
depth and specificity of the etch may be varied by the etching
technique chosen so that microwells of the desired volume may be
formed. In certain embodiments, the etching process creates
microwells by preferentially etching the core material of the
individual glass fibers in the bundle such that each well is
approximately aligned with a single fiber and isolated from
adjacent wells by the cladding material. Potential advantages of
the fiber optic array format is that it can produce thousands to
millions of reaction vessels without complicated microfabrication
procedures and that it can provide the ability to observe and
optically address many reaction vessels simultaneously. An example
of an etched fiber optic array is shown in FIGS. 13A and 13B. FIG.
13A shows a fiber optic array that has been etched to form a
plurality or reaction vessels. In this particular example, the
wells have a diameter of approximately 4.5 um and a volume of about
46 fL. FIG. 13B shows an AFM image of a portion of the fiber optic
array of FIG. 13A.
[0132] Each microwell may be aligned with an optical fiber in the
bundle so that the fiber optic bundle can carry both excitation and
emission light to and from the wells, enabling remote interrogation
of the well contents. Further, an array of optical fibers may
provide the capability for simultaneous or non-simultaneous
excitation of molecules in adjacent vessels, without signal
"cross-talk" between fibers. That is, excitation light transmitted
in one fiber does not escape to a neighboring fiber.
[0133] In certain embodiments of the present invention, the
physical alterations to a fiber optic may be made as taught in U.S.
Pat. Nos. 6,023,540, 6,327,410, and 6,858,394. Any one or more of
the surface of the glass microwells, the surface of the sealing
component, or particles within microwells can be functionalized in
certain embodiments to create binding surface(s).
[0134] Alternatively, the equivalent structures can be fabricated
using other methods that do not utilize the ends of an optical
fiber bundle as a substrate. For example, the array may be a
spotted, printed or photolithographically fabricated substrate
produced by techniques known in the art; see for example
WO95/25116; WO95/35505; PCT US98/09163; U.S. Pat. Nos. 5,700,637,
5,807,522, 5,445,934, 6,406,845, and 6,482,593. In some cases, the
array may be produced using molding and/or etching techniques as
will be known to those of ordinary skill in the art.
[0135] In certain embodiments, the present invention provides a
system equipped with a mechanical platform that applies a sealing
component to a substrate. The platform may be positioned beneath a
stage on the system. After the chosen reaction components have been
added to an array of reaction vessels, the sealing component may be
mated with the array. For example, the sealing component may be
sandwiched between a flat surface (such as, for example, a
microscope slide) and the array of reaction vessels using uniform
pressure applied by the mechanical platform.
[0136] A non-limiting embodiment is illustrated in FIG. 14A. A
sealing component 300 is placed on top of mechanical platform 302.
The fluid sample 304 is placed on top of the sealing component 300.
The mechanical platform is moved upwards towards the array 306
(e.g., fiber optic array) such that uniform pressure is applied. As
shown in FIG. 14B, the sealing component 300 forms a tight seal
with the array 306. In other instances, varying pressure may be
applied to the sealing component to form a tight seal between the
sealing component and the array. The system may also comprise
additional components 312 that may be utilized to analyze the array
(e.g., microscope, computer, etc.) as discussed more herein.
[0137] In some embodiments, the first and/or second substrate to
which the analyte molecules/dissociated species are immobilized may
be subjected to at least one washing step. In one instance, a
substrate may be washed after exposing the substrate to one or more
solutions comprising analyte molecules, binding ligands, precursor
labeling agent molecules, or dissociated species. In some
instances, the wash step(s) may be used to wash away any analyte
molecules or non-analyte molecules, any binding ligands or
non-binding ligands, any precursor labeling agent molecules or
non-precursor labeling agent molecules, or any dissociated species
or non-dissociated species that are not immobilized with respect to
the substrate. The wash step(s) may be performed by any suitable
technique known to those of ordinary skill in the art, for example,
by submersion of the substrate in a wash solution, flushing the
substrate with a wash solution, etc. In certain embodiments, the
wash solution is selected so that it does not cause appreciable
change to the configuration of the substrate surface and/or does
not disrupt an interaction between at least two components of the
assay (e.g., a capture component and an analyte
molecule/dissociated species). In other cases, the wash solution
may be a solution that is selected to chemically interact with one
or more components of the substrate. As will be understood by those
of ordinary skill in the art, a wash step may be performed at any
appropriate time point during the inventive methods (e.g., after
exposure of the substrate to a reagent or after immobilization of
and agent with respect to a substrate) during a method of the
present invention.
Exemplary Target Analytes
[0138] As will be appreciated by those in the art, a large number
of analyte molecules and particles may be detected and, optionally,
quantified using methods and systems of the present invention;
basically, any analyte molecule or particle that is able to be made
to become immobilized with respect to (e.g., by binding) a capture
component can be potentially investigated using the invention.
Certain more specific targets of potential interest that may
comprise an analyte molecule or particle are mentioned below. The
list below is exemplary and non-limiting.
[0139] In some embodiments, the analyte molecule may be an enzyme.
Non-limiting examples of enzymes include, an oxidoreductase,
transferase, kinase, hydrolase, lyase, isomerase, ligase, and the
like. Additional examples of enzymes include, but are not limited
to, polymerases, cathepsins, calpains, amino-transferases such as,
for example, AST and ALT, proteases such as, for example, caspases,
nucleotide cyclases, transferases, lipases, enzymes associated with
heart attacks, and the like. When a system/method of the present
invention is used to detect the presence of viral or bacterial
agents, appropriate target enzymes include viral or bacterial
polymerases and other such enzymes, including viral or bacterial
proteases, or the like.
[0140] In other embodiments, the analyte molecule or particle may
comprise an enzymatic component. For example, the analyte particle
can be a cell having an enzyme or enzymatic component present on
its extracellular surface. Alternatively, the analyte particle is a
cell having no enzymatic component on its surface. Such a cell is
typically identified using an indirect assaying method described
below. A non-limiting example of an enzymatic component is
beta-galactosidase.
[0141] In yet other embodiments, the analyte molecule may be a
biomolecule. Non-limiting examples of biomolecules include
hormones, antibodies, cytokines, proteins, nucleic acids, lipids,
carbohydrates, lipids cellular membrane antigens and receptors
(neural, hormonal, nutrient, and cell surface receptors) or their
ligands, or combinations thereof. Non-limiting embodiments of
proteins include peptides, polypeptides, protein fragments, protein
complexes, fusion proteins, recombinant proteins, phosphoproteins,
glycoproteins, lipoproteins, or the like. As will be appreciated by
those in the art, there are a large number of possible
proteinaceous analyte molecules that may be detected or evaluated
for binding partners using the present invention. In addition to
enzymes as discussed above, suitable protein analyte molecules
include, but are not limited to, immunoglobulins, hormones, growth
factors, cytokines (many of which serve as ligands for cellular
receptors), cancer markers, etc.
[0142] In certain embodiments, the analyte molecule may be a
host-translationally modified protein (e.g., phosphorylation,
methylation, glycosylation) and the capture component may be an
antibody specific to a post-translational modification. Modified
proteins may be captured with capture components comprising a
multiplicity of specific antibodies and then the captured proteins
may be further bound to a binding ligand comprising a secondary
antibody with specificity to a post-translational modification.
Alternatively, modified proteins may be captured with capture
components comprising an antibody specific for a post-translational
modification and then the captured proteins may be further bound to
binding ligands comprising antibodies specific to each modified
protein.
[0143] In another embodiment, the analyte molecule is a nucleic
acid. A nucleic acid may be captured with a complementary nucleic
acid fragment (e.g., an oligonucleotide) and then optionally
subsequently labeled with a binding ligand comprising a different
complementary oligonucleotide.
[0144] Suitable analyte molecules and particles include, but are
not limited to small molecules (including organic compounds and
inorganic compounds), environmental pollutants (including
pesticides, insecticides, toxins, etc.), therapeutic molecules
(including therapeutic and abused drugs, antibiotics, etc.),
biomolecules (including hormones, cytokines, proteins, nucleic
acids, lipids, carbohydrates, cellular membrane antigens and
receptors (neural, hormonal, nutrient, and cell surface receptors)
or their ligands, etc), whole cells (including prokaryotic (such as
pathogenic bacteria) and eukaryotic cells, including mammalian
tumor cells), viruses (including retroviruses, herpesviruses,
adenoviruses, lentiviruses, etc.), spores, etc.
[0145] The fluid sample comprising or suspected of comprising and
analyte molecule or particle may be derived from any suitable
source. In some cases, the sample may comprise a liquid, fluent
particulate solid, fluid suspension of solid particles,
supercritical fluid and/or gas. In some cases, the analyte molecule
or particle may be separated or purified from its source prior to
determination; however, in certain embodiments, an untreated sample
containing the analyte molecule or particle may be tested directly.
The source of the analyte molecule may be synthetic (e.g., produced
in a laboratory), the environment (e.g., air, soil, etc.), a
mammal, an animal, a plant, or any combination thereof. In a
particular example, the source of an analyte molecule is a human
bodily substance (e.g., blood, serum, plasma, urine, saliva,
tissue, organ, or the like).
Binding Ligands and Precursor Labeling/Labeling Agent Molecules
[0146] Binding ligands may be selected from any suitable molecule,
particle, or the like, as discussed more below, able to associate
with an analyte molecule and/or to associate with another binding
ligand. For example, when only a first binding ligand is employed,
a first binding ligand may associate with an analyte molecule. In
another example, when a first binding ligand and a second binding
ligand are employed, a first binding ligand may associate with an
analyte molecule and a second binding ligand may become immobilized
with respect to the analyte molecule by becoming associated with
the first binding ligand. Some non-limiting examples of potentially
suitable binding ligands are discussed more below.
[0147] Certain binding ligands can comprise a component that is
able to facilitate detection, either directly or indirectly. A
component may facilitate indirect detection, for example, by
converting a precursor labeling agent molecule to a labeling agent
molecule (e.g., an agent that is detected in an assay). In some
embodiments, the binding ligand may comprise an enzymatic component
(e.g., horseradish peroxidase, beta-galactosidase, etc). A first
binding ligand may or may not be used in conjunction with
additional (e.g., second, etc.) binding ligands, examples of which
are discussed herein.
[0148] A "precursor labeling agent" is any molecule, particle, or
the like, that can be converted to a labeling agent molecule upon
exposure to a suitable converting agent. Such a converting agent
may, in certain embodiments, comprise at least a portion of at
least one of a capture component, an analyte, and/or a binding
ligand. In certain such embodiments, it is advantageous if the
converting agent comprises at least a portion of the dissociated
species released from the first substrate and transferred to the
second substrate for detection. A "labeling agent" is any molecule,
particle, or the like, that facilitates detection, by acting as the
detected entity, using a chosen detection technique.
[0149] In some embodiments, binding ligands may be selected such
that are able to function in the following manner. If only a first
binding ligand is employed, the first binding ligand may associate
directly with an analyte molecule. In other cases, if both a first
and a second binding ligand are employed, the first binding ligand
may associate with an analyte molecule and comprise a component
which is able to interact with the second binding ligand. Certain
assays may also comprise additional components (e.g., a third
binding ligand that can interact with the second binding ligand,
first binding, or analyte). In certain embodiments, any one or more
of the binding ligands may comprise at least a portion thereof able
to convert a precursor labeling agent into a labeling agent
molecule. In some cases, an assay component may be able to
associate with more than one other assay component, for example, a
third binding ligand may associate with both a first and a second
binding ligand.
[0150] As mentioned above, in certain embodiments, a first binding
ligand may be employed alone or in conjunction with a second (and a
third, etc.) binding ligand. A first binding ligand may be selected
(e.g., in the case when only a first and a second binding ligand is
employed) such that it may associate with an analyte molecule and
be able to interact with the second binding ligand (e.g., such that
the first binding ligand is associated with the second binding
ligand).
[0151] In some embodiments, the substrate (e.g., the substrate
in/on which the plurality of analyte molecules are immobilized) may
be exposed to a plurality of first binding ligands such that a
first binding ligand associates with substantially all of the
plurality of analyte molecules or particles to form a plurality of
immobilized complexes. In some cases, the substrate may also be
exposed to a plurality of second binding ligands such that the
second binding ligand associates with substantially all of the
plurality of first binding ligands which are associated with an
analyte molecule or particle to form a plurality of immobilized
complexes.
[0152] In yet other cases, the substrate may be additionally
exposed to a plurality of third binding ligands such that a third
binding ligand associates with substantially all of the second
binding ligands which are associated with a first binding ligand
which are associated with an analyte molecule or particle to form
at least a portion of the plurality of immobilized complexes.
Substantially all, as used herein, refers to at least greater than
80%, greater than 85%, greater than 90%, greater than 95%, greater
than 97%, greater than 98%, greater than 99%, greater than 99.5%,
or more, of a molecule/particle/species of a specific type.
[0153] As discussed more below, a binding ligand (e.g., a first,
second and/or third, etc. binding ligand) may comprise a cleavable
linker (e.g., disulfide). In some cases, at least one of the
binding ligands may comprise biotin.
[0154] In some cases, binding ligands of the present invention may
be functionalized to reduce non-specific binding, e.g., direct
binding or association of a binding ligand to another component
(e.g., capture component, surface of a substrate, etc.) of the
assay, where such binding is undesired. In certain such
embodiments, the binding ligand may be functionalized with a large
steric molecule and/or particle which prevent the binding ligand
from approaching and/or associating with the substrate, another
component, etc. For example, the binding ligand may be
functionalized with poly(ethylene glycol) groups or particles,
poly(ethylene oxide) groups or particles (e.g., particles which are
protein resistant such as beads coated in poly(ethylene glycol),
DNA, or zwitterionic lipids).
Dissociation/Release of Immobilized Complexes/Species
[0155] In some embodiments, a plurality of dissociated species is
formed during the assay. A "dissociated species," as used herein,
is a molecule, complex, or particle (which, as previously noted,
includes biological cells and sub cellular organelles) at least a
portion of which has been released/dissociated from a molecule,
complex, particle, surface, etc., to which it was immobilized. The
dissociated species may form by causing an association (e.g., bond,
attachment, etc.) to cleave, thereby releasing the dissociated
species. A molecule or particle may be released from a substrate in
certain instances without causing cleavage of a bond (e.g., by
physical means); such released molecules or particles may still be
referred to herein as dissociated species.
[0156] The plurality of dissociated species, in some cases, may be
formed by the dissociation of at least a portion of a plurality of
complexes immobilized with respect to a substrate. Each immobilized
complex, in most cases, will comprise an analyte molecule, a
capture component, and, in some cases, at least one binding ligand.
In some cases, each immobilized complex may additionally comprise
at least one cleavable linker (e.g., a disulfide linker) and/or an
enzymatic component. A dissociated species, therefore, may comprise
any portion of the previously immobilized complex (e.g., at least a
portion of any one or more of the analyte molecule, the capture
component and/or the at least one binding ligand). For example, at
least one or each of the plurality of dissociated species may
comprise at least a portion of a capture component, analyte
molecule, and/or binding ligand(s) (e.g., first, second, third,
etc., binding ligands).
[0157] The number of dissociated species released from the
substrate may be, but need not be, substantially equal to the total
number of analyte molecules or particles in the fluid sample and/or
immobilized on the substrate. For example, in certain embodiments,
the ratio of immobilized analyte molecules to dissociated species
may be about 1:1, about 2:1, about 5:1, about 10:1, about 100:1,
about 1000:1, about 10000:1, or the like. In some cases, the ratio
is greater than about 1:1 but does not exceed 10:1, greater than
about 10:1 but does not exceed 100:1, greater than about 100:1 but
does not exceed 1000:1, greater than about 1000:1 but does not
exceed 10000:1, greater than about 10000:1 but does not exceed
1,000,000:1, or greater than about 1,000,000:1 in certain
embodiments. In some embodiments, the number of the plurality of
immobilized molecules or particles released is approximately equal
to, is directly proportional to, is about one half of, is about one
quarter of, is about one tenth of, is about one hundredth of, is
about one thousandth of, is about one ten-thousandth of, or is
about one hundred-thousandth of, the number of the plurality of
analyte molecules or particles captured.
[0158] A dissociated species, in some cases, may comprise a
component which is able to be detected, either indirectly or
directly (i.e., may comprise a labeling agent molecule and/or is
capable of converting a precursor labeling agent molecule to a
labeling agent molecule). The presence of a detectable component on
a dissociated species can allow for the detection of the
dissociated species, thereby allowing for the determination and/or
quantification of the dissociated species. The determination and/or
quantification of the dissociated species, can be related to the
detection and/or quantification of the analyte molecule in a fluid
sample, as discussed more below. In a particular embodiment, the
detectable component is an enzymatic component.
[0159] In some embodiments, at least a portion of at least some of
the immobilized complexes may be susceptible to being dissociated
from a substrate by exposure to a dissociating agent, thereby
forming a plurality of dissociated species. As mentioned above,
non-limiting examples of dissociating agents include chemical
dissociating agents, electromagnetic radiation, a change in the pH,
a change in the temperature, etc. In a particular example, exposing
the plurality of immobilized complexes to a reducing agent causes
at least a portion of at least some of the plurality of complexes
to dissociate from the substrate to form a plurality of dissociated
species.
[0160] In some cases, the dissociating agent may have essentially
no specific affinity for the capture components. That is, the
dissociating agent does not cause the dissociating species to form
by interacting with the capture component and employing competitive
binding to release the analyte molecule that associated with the
capture component. In some cases, when the dissociating agent has
essentially no specific affinity for the capture component, the
dissociated species may comprise an enzymatic component.
[0161] In some cases, each of the plurality of immobilized
complexes may comprise a cleavable linkage and the plurality of
dissociated species may be formed by cleaving at least some of the
cleavable linkages. In some instances, a dissociating agent may be
employed in conjunction with a cleavable linkage, wherein exposure
of the cleavable linkage to the dissociating agent causes the
cleavable linkage to cleave and a dissociated species to form. As
mentioned above, a cleavable linkage is a linkage that is able to
be cleaved upon exposure to a dissociating agent. The cleavable
linkage may be present in any portion of the immobilized complex
(e.g., the analyte molecule, the binding ligand (e.g., first,
second or third, etc. binding ligand), the capture component,
etc.). In some embodiments, the cleavable linkage may be a linkage
that is specifically cleaved, that is, a linkage that can be
cleaved without altering or damaging any other portion of the
complex not being cleaved and which relatively uniformly alters the
molecule in a reproducible manner. It should be understood, that a
cleavable linkage may be selected depending on various parameters
including desired size, solubility, ability to cleave or not cleave
the linkage under certain conditions, the type of cleavage method
to be used, etc. A cleavable linkage may be homobifunctional (e.g.,
contain two or more identical leaving groups) or heterobifunctional
(e.g., contain two or more different leaving groups).
[0162] In some cases, the cleavable linkage may be a photocleavable
linkage and the plurality of dissociated species may be formed by
exposing the substrate to electromagnetic radiation (e.g.,
ultraviolet light). In certain such instances, the photocleavable
linkage may be cleaved at a wavelength of light that does not
damage the molecule being released, for example, in the ultraviolet
to visible range. Non-limiting examples of photocleavable linkages
include linkages containing o-nitrobenzyl, desyl,
trans-o-cinnamoyl, m-nitrophenyl, benzylsulfonyl groups and
others.
[0163] In some embodiments, the cleavable linkage may be a
chemically cleavable linkage (i.e., susceptible to cleavage upon
exposure to a chemical dissociating agent) Non-limiting examples of
chemically cleavable linkages which may be cleaved by a chemical
dissociating agent include a disulfide, which can be cleaved with
reducing agents; a diol, which can be cleaved with periodates; a
diazo bond, which can be cleaved with dithionates; an ester, which
can be cleaved with hydroxylamines; a sulfone, which can be cleaved
with bases, and others known in the art, as discussed more below.
The concentration of dissociating agent used to bring about
dissociation will depend on the type of cleavable linkage and
dissociating agent employed as well as other assay conditions. The
appropriate concentration and conditions to bring about a desired
degree of dissociation may be selected in particular applications
using routine screening tests and optimization. For example, in
certain particular applications, the immobilized complexes may be
exposed to a chemical dissociating agent in a concentration of
between about 10 mM and about 100 mM, between about 1 mM and about
1 M, between about 0.1 M and about 1 M, etc.
[0164] In some embodiments, the plurality of dissociated species is
formed by exposing the substrate to a chemical dissociating agent.
The chemical dissociating agent may comprise at least one of a pH
altering agent, an ionic strength altering agent, a denaturing
agent, a reducing agent, a reactive agent, or an enzyme. In a
particular embodiment involving immobilized complexes comprising
thiol linkages, the chemical dissociating agent may comprise
beta-mercaptoethanol. The plurality of complexes may be exposed to
the chemical dissociating agent thereby forming a plurality of
dissociated species. In some cases, a chemical dissociating agent
is particularly suited to cleave a particular cleavable linkage.
Non limiting combinations of chemical dissociating agents and
cleavable linkages will now be discussed in more detail.
[0165] In some embodiments, the chemical dissociating agent is a
reducing agent. A reducing agent is a compound or complex that
chemically reduces another chemical species suseptible to chemical
reduction which can, in certain cases, lead to scission of the
species. In the case of a chemical dissociating agent that is a
reducing agent, the immobilized complex may be reduced by the
reducing agent and at least a portion of the complex may be
dissociated by the reducing agent to form a dissociated species.
Non-limiting examples of reducing agents that may be used to cleave
a cleavable linkage include sodium borohydride,
beta-mercaptoethanol, cysteine, tris(2-carboxyethyl)phosphine, zinc
and dilute acid, triphenylphosphine (Ph.sub.3P) and water,
dithiothreitol (DTT), and others known in the art.
[0166] In some embodiments, a chemical dissociating agent may be
substantially removed from the solution comprising the plurality of
dissociated species prior to further manipulation of the plurality
of dissociated species (e.g., partitioning of the plurality of
dissociated species across a plurality of reaction vessels). For
example, the chemical dissociating agent may be removed by
filtration through a material to which the chemical dissociating
agent may bind, by precipitation, or the like. In other
embodiments, the chemical dissociated may not be substantially
removed prior to further manipulation of the solution comprising
the plurality of dissociated species.
[0167] In some embodiments, each immobilized complex may comprise a
disulfide bond, some of which may be cleaved to form a plurality of
dissociated species. In some cases, each immobilized complex may
comprise at least one capture component, at least one analyte
molecule or particle, and at least one disulfide linkage. At least
some of the disulfide linkages may be cleaved to form a plurality
of dissociated species (e.g., which are not immobilized with
respect to the substrate). Techniques for formation of a disulfide
bond in a component of the immobilized complex (e.g., as part of
the capture component, the analyte molecule, and/or a binding
ligand(s)) are known to those of ordinary skill in the art. A
non-limiting reagent that may be used for the formation of a
disulfide linkage is pyridyl disulfide-containing
heterobifunctionals (e.g., SDPD,
N-succinimidyl-pyridyldithioproprionate and SMPT,
4-succinimidyloxycarbonyl-.alpha.-methyl-.alpha.-(2-pyridyldith-
io)toluene). Disulfide bonds may be cleaved, for example, through
hydrogenolysis by a reducing agent (e.g., beta-mercaptoethanol,
dithiothreitol, dithioerythritol, cysteine,
tris(2-carboxytheyl)phosphine, and/or butanethiol) or through a
disulfide interchange process with a compound containing one or
more free sulfhydryls.
[0168] A disulfide linkage may be cleaved in certain embodiments by
exposing the linkage to a reducing agent. In some cases, the
reduction will yield unconjugated molecules, one or both of which
may contain a portion of the disulfide linkage, and in some cases,
a free sulfhydryl (e.g., --SH) will be formed on one or both the
ends of the cleaved linkage. A non-limiting example of a how a
disulfide linkage may be cleaved is shown in Equation 1:
##STR00001##
wherein X and Y are components of the immobilized complex relative
to the disulfide linkage. A chemical reducing agent reacts with the
disulfide bonds to generate two free sulfhydryl groups per original
disulfide bond.
[0169] In a particular embodiment of the present invention, a
binding ligand may comprise a disulfide linkage. The disulfide
group may be cleaved by exposure of an immobilized complex
comprising the binding ligand to beta-mercaptoethanol. In other
embodiments, a capture component may comprise a disulfide linkage
that may be cleaved by exposure to beta-mercaptoethanol and/or
another reducing agent.
[0170] In other embodiments, the cleavable linkage may be a diol or
a glycol. A diol or glycol linkage may be incorporated into a
portion of the immobilized complex using methods that are known to
those skilled in the art (e.g., inclusion of a tartaric acid
residue). In some cases, the glycol or diol linkage may be cleaved
by oxidation. For example, the immobilized complex comprising a
diol or a glycol cleavable linkage may be exposed to a reagent such
as sodium periodate or palladium acetate (Pd(OAc).sub.2), and the
linkage may be cleaved to form aldehydes. A non-limiting example of
the cleavage of a glycol linkage is shown in Equation 2:
##STR00002##
wherein X and Y are components of the immobilized complex relative
to the glycol linkage.
[0171] In yet other embodiments, the cleavable linkage may be a
diazo bond. A diazo bond may be incorporated into a portion of the
immobilized complexes using methods that are known to those skilled
in the art (e.g., coupling between an arendiazonium cation and a
phenol). For non-limiting examples see March, J.; Advanced Organic
Chemistry, New York, John Wiley and Sons, 1992, pages 591-592, 638,
etc. A diazo linkage may be cleaved upon exposure to a chemical
dissociating agent such as dithionite, zinc metal, and sodium
hydrosulfite (Na.sub.2S.sub.2O.sub.4) to form primary amines. A
non-limiting example of the cleavage of a diazo linkage is shown in
Equation 3:
##STR00003##
wherein X and Y are components of the immobilized complex relative
to the diazo linkage.
[0172] In yet other embodiments, the linkage may be an ester or a
thioester. An ester or a thioester linkage may be cleaved upon
exposure to a nucleophile, such as hydroxylamine. The cleavage of
an ester with hydroxylamine may result in the formation of an amide
derivative on one side of the cleaved crosslinker and a hydroxyl
group on the other side, as shown in Equation 4.
##STR00004##
wherein X and Y are the parts of the immobilized complex relative
to the ester linkage.
[0173] In still yet other embodiments, the linkage may be a sulfone
linkage. Sulfone linkages that may be incorporated into a complex
include 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone (DFDNPS) and
bis(2-[succinimidooxycarbonyloxy]ethyl)sulfone (BSOCOES). The
sulfone linkage may be cleaved by hydrolysis under basic
conditions, such as in the presence of sodium phosphate. The
cleavage of a sulfone group is shown in Equation 5:
##STR00005##
wherein X and Y are components of the immobilized complex relative
to the sulfone linkage.
[0174] In other cases, the cleavable linkage may be an
enzymatically cleavable linkage. For example, a protease may be
used to cleave a cleavable functional group having a suitable
recognition sequence for the protease. Particularly useful
proteases are endopeptidases such as factor Xa, tobacco etch virus
(TEV) protease, trypsin, chymotrypsin, Staphylococcus aureus
protease, submaxillaris protease, and others. The protease can be
selected based on the incorporation of a particular cleavable
recognition sequence as a functional group. Other considerations
for selecting a protease include the presence or absence of a
recognition sequence in the molecule being captured and
released.
[0175] In yet other cases, the cleavable linkage may be a
pH-cleavable linkage and comprise pH-labile bonds. For example,
ketals may be labile (e.g., cleaved) in acidic environments (e.g.,
pH less than 7) and form a diol and a ketone. As another example,
acetals may be labile (e.g., cleaved) in acidic environments (e.g.,
pH less than 7) and form a diol and an aldehyde. As yet another
example, imines or iminiums may be labile in acidic environments
(e.g., pH less than 7) and form an amine and an aldehyde or a
ketone, respectively. In other instances, a cleavable linkage may
be cleaved in basic conditions (e.g., sulfone linkage).
[0176] In some embodiments, a method of the present invention may
comprise releasing a plurality of molecules or particles from the
first substrate. The plurality of molecules or particles released
from the first substrate may comprise at least a portion of an
analyte molecule or particle captured on the first substrate or may
be free of any portion of an analyte molecule or particle captured
on the first substrate. A plurality of molecules or particles may
be released by exposing the substrate to electromagnetic radiation
(e.g., ultraviolet light). As another example, a plurality of
molecules or particles may be released by exposing the substrate to
a chemical dissociating agent (e.g., beta-mercaptoethanol). As yet
another example, a plurality of molecules or particles may be
released by physical manipulations (e.g., scraping the substrate,
agitation, etc.).
Detection
[0177] The plurality of dissociated species may be detected and/or
quantified, and the detection and/or quantification may be related
to the presence and, optionally, quantity and/or concentration of
analyte molecules/particles in the sample being tested. In some
embodiments, the plurality of dissociated species may be detected
and/or quantified by partitioning the plurality of dissociated
species across a plurality of reaction vessels (e.g., in an array).
In certain embodiments, the partitioning may occur such that at
least some (e.g., a statistically significant fraction) of the
reaction vessels comprise at least one or, in certain cases, one
dissociated species and at least some (e.g., a statistically
significant fraction) of the reaction vessel comprises no
dissociated species. In some cases, a detector may be configured to
detect dissociated species in or on a substrate (e.g., an array of
reaction vessels).
[0178] In some embodiments, when a first substrate is used to
capture a plurality of analyte molecules and/or particles and a
second substrate is used to detect a plurality of dissociated
species, the number of dissociated species detected on or within
the second substrate may be directly proportional to the number of
dissociated species released from the second substrate. In other
embodiments, the number of dissociated species detected on or
within the second substrate may be approximately equal to, less
than about one half, less than about one quarter, less than about
one tenth, less than about one hundredth, less than about one
thousandth, less than about one ten-thousandth, or the like, of the
number of the plurality of dissociated species dissociated from the
first substrate.
[0179] In some embodiments, the number of dissociated species
detected is no greater than the number of dissociated species
released and/or the number of dissociated species is no greater
than the number of analyte molecules or particles associated with a
first substrate. For example, the number of dissociated species
detected (e.g., on a second substrate) may be approximately equal
to, no greater than about three quarters of, no greater than about
one half of, no greater than about one quarter of, no greater than
about one tenth of, no greater than about one hundredth of, no
greater than about one thousandth of, no greater than about ten
thousandth of, or no greater than about one hundred thousandth of,
the number of analyte molecules or particles associated (e.g.,
captured) with a first substrate and/or the number of dissociated
species released (e.g., from a first substrate).
[0180] The dissociated species may be detected and/or quantified
directly or indirectly. In the case or direct detection, the
dissociated species itself comprises a molecule or moiety that may
be directly interrogated and detected, for example, a fluorophore.
A reaction vessel that comprises such a dissociated species will
therefore emit a signal upon interrogation of the reaction
vessel.
[0181] The indirect approach can include, for example, exposing
dissociated species comprising at least one precursor labeling
agent molecule to a suitable converting agent to produce a
detectable labeling agent molecule. In other embodiments, the
dissociated species may comprise a converting agent and may be
exposed within the reaction vessel to a precursor labeling agent
molecule to produce a detectable labeling agent molecule. In some
cases, at least one precursor labeling agent molecule is converted
to a labeling agent molecule when contained in a reaction vessel
comprising a dissociated species. The presence or absence of a
dissociated species in a reaction vessel may then be determined by
determining the presence or absence of a labeling agent molecule in
the reaction vessel. For example, the dissociated species may
comprise an enzymatic component and the precursor labeling agent
molecule may be a chromogenic, fluorogenic, or chemiluminescent
enzymatic precursor labeling agent molecule. Exposure of the
precursor labeling agent molecule to the enzymatic component may
convert the precursor labeling agent molecule to a chromogenic,
fluorogenic, of chemiluminescent labeling agent molecule that may
be detected. In another instances, the indirect approach can
include, for example, the situation where the dissociated species
does not have intrinsic activity to convert a precursor labeling
agent molecule to a labeling agent molecule and is not detectable
itself. Therefore, the dissociated species may be exposed to at
least one dissociated species binding ligand which may associate
with the dissociated species, thus forming a complex comprising the
dissociated species and the dissociated species binding ligand. The
dissociated species binding ligand may comprise a component that is
able to convert a precursor labeling agent molecule into a labeling
agent molecule or may comprise a precursor labeling agent molecule
able to be converted by exposure to a converting agent. Thereafter,
the presence of the labeling agent molecule can be determined to
determine whether a dissociated species is present in a reaction
vessel. It is to be understood that a variety of dissociated
species, dissociated species binding ligands, and precursor
labeling agent molecules, as will be discussed in more detail
below, may be used in the practice of the invention. To the extent
that a dissociated species comprises an enzymatic component,
enzymatic precursor labeling agent molecules can be added to the
reaction vessel whereupon the enzymatic component converts the
precursor labeling agent molecule into a chromogenic, fluorogenic,
or chemiluminescent labeling agent molecule and permits the
detection of the dissociated species.
[0182] Two non-limiting examples of indirect detection of a
dissociated species are illustrated in FIGS. 15A and 15B. In FIG.
15A, a reaction vessel 30 is provided which comprises a detection
capture component 32. Dissociated species 34 is associated with
detection capture component 34. The reaction vessel is exposed to
precursor labeling agent molecule 36, which upon exposure to
dissociated species 34 carrying a converting agent, converts to a
labeling agent molecule 38, as indicated by arrow 37. As another
example, in FIG. 15B, a reaction vessel 40 is provided which
comprises a detection capture component 42. Dissociated species 44
is associated with the detection capture component 42. A
dissociated species binding ligand 46 is provided which associates
with dissociated species 44. The reaction vessels is exposed to
precursor labeling agent molecule 48 which upon exposure to
dissociated species binding ligand 44 converts to labeling agent
molecule 50, as indicated by arrow 49.
[0183] In some embodiments, a dissociated species may comprise an
enzymatic component (e.g., from a binding ligand). In this
instance, the precursor labeling agent molecule may be an enzymatic
label, for example, a chromogenic, fluorogenic, or chemiluminescent
enzymatic precursor labeling agent molecule, that upon contact with
the enzymatic component, converts to a labeling agent molecule. In
some cases, the chromogenic, fluorogenic, or chemiluminescent
enzymatic precursor labeling agent molecule is provided in an
amount sufficient to contact every dissociated species which was
partitioned across a plurality of reaction vessels. In some cases,
the enzymatic component may comprise beta-galactosidase or
horseradish peroxidase.
[0184] As will be understood by those of ordinary skill in the art,
chromogenic, fluorogenic, or chemiluminescent enzymatic precursor
labeling agent molecules may be selected for conversion by many
different enzymes. Thus, any known chromogenic, fluorogenic, or
chemiluminescent enzyme precursor labeling agent molecule capable
of producing a labeling agent molecule in a reaction with a
particular enzyme can potentially be used in the present invention
as the precursor labeling agent molecule in embodiments where the
dissociated species comprises an enzymatic component (or a
dissociated species binding ligand comprises an enzymatic
component). For example, many chromogenic, fluorogenic, or
chemiluminescent precursor labeling agent molecules suitable for
use an enzymatic precursor labeling agent molecule are disclosed in
The Handbook--A Guide to Fluorescent Probes and Labeling
Technologies, Tenth Ed., Chapter 10.
[0185] In some embodiments, the plurality of reaction vessels may
additionally comprise at least one dissociated species capture
component. A "dissociated species capture component" is a capture
component that specifically binds to or otherwise captures a
dissociated species, such that the dissociated species is
immobilized during the assay. Generally, the dissociated species
capture component allows the attachment of a dissociated species to
a solid support (that is, the surface of a microwell, a sealing
component or a nanoparticle) for the purposes of detection,
quantification, or other analysis. In some cases, a method of the
present invention will comprise the step of immobilizing at least
one of the plurality of dissociated species with respect to the at
least one dissociated species capture component.
[0186] In some embodiments, at least some of the plurality of
dissociated species may be immobilized with respect to at least one
particle comprising at least one binding partner for the
dissociated species, thereby forming a plurality of complexes. A
plurality of particles comprising a binding partner may be
provided, in some cases, such that one or zero dissociated species
released from the substrate associates with each binding partner of
at least a fraction of the plurality of particles comprising a
binding partner. The particles may have an average diameter of less
than about 100 um, less than about 50 um, less than about 10 um,
less than about 1 um, less than about 500 nm, less than about 100
nm, less than about 10 nm, less than about 1, or the like. In some
cases, the particles may be provided such that there is greater
than about 10, greater than about 100, greater than about 1000,
greater than about 10,000, greater than about 100,000, greater than
about 1,000,000, or the like, particles provided for every
dissociated species released from the substrate. In some such
embodiments, the plurality of dissociated species may be
immobilized with respect to a plurality of particles prior to
partitioning across a plurality of reaction vessels. In certain
embodiments, the plurality of dissociated species may be exposed to
a plurality of particles such that either one or no dissociated
species are immobilized with respect to a single particle. The
particles may comprise a plurality of binding partners (e.g.,
biotin) with respect to which a dissociated species may be
immobilized, thereby forming a plurality of particle-containing
complexes. The particle-containing complexes may be partitioned
across a plurality of reaction vessels such that at least some
(e.g., a statistically significant fraction) of the plurality of
reaction vessels contain zero particle-containing complexes and at
least some (e.g., a statistically significant fraction) of the
plurality of reaction vessels contain only one or at least one
particle-containing complex. The reaction vessels which comprise a
particle-containing complex may then be determined, and the methods
describe herein may be used to detect and/or quantify the
dissociated species and analyte molecules. For example, the number
or fraction of the plurality of reaction vessels that contain a
particle-containing complex may be related to a measure of the
concentration of analyte molecules or particles in the fluid
sample. The particle-containing complexes may be detected directly
or indirectly using methods as discussed herein (e.g.,
electromagnetic radiation, microscopy, etc.). Each reaction vessel
may comprise at least about 10 particles, at least about 100
particles, at least about 500 particles, at least about 1000
particles, at least about 5000 particles, at least about 10000
particles, or the like, wherein in some reaction vessels, at least
one particle may comprise a complex (e.g., a particle-containing
complex). Each reaction vessel may comprise approximately the same
or differing number of particles as the reaction vessels which it
is adjacent to.
[0187] A non-limiting embodiment of the above assay method is
depicted in FIG. 16. A plurality of particles 330 are exposed to a
plurality of dissociated species such that at least some of each of
the plurality of dissociated species 332 are immobilized with
respect to a particles 330 to form a plurality of dissociated
species in the form of particle-containing complexes 334 (step
(A)). The plurality of particles and particle-containing complexes
may be partitioned across a plurality of reaction vessels 336 such
that at least some of the plurality of reaction vessels contain
only one or at least one particle-containing complex 338 and at
least some of the plurality of reaction vessels will contain zero
particle-containing complexes 340 (step (B)).
[0188] In some embodiments, at least a fraction of the number of
dissociated species may be detected substantially simultaneously.
"Substantially simultaneously" when used in conjunction with
detection, as used herein, refers to detection of the
species/molecules/particles of interest at approximately the same
time, as opposed to sequentially detected. A plurality of
species/molecules/particles may be detected substantially
simultaneously using various techniques, including optical
techniques (e.g., CCD detector). In some embodiments involving
substantially simultaneous detection, each of the detected
species/molecules/particles (e.g., dissociated species) is
spatially separated with respect to the other detected
species/molecules/particles (e.g., dissociated species) during
detection, such that detection is able to resolve individual
dissociated species of the plurality of dissociated species
detected. For example, resolving individual molecules/particles of
a plurality of molecules/particles that are partitioned across a
plurality of reaction vessels such that each reaction vessel
contains zero or only one species/molecule/particle involves
detecting molecules/particles that are spatially separated. In some
cases, at least about 80%, at least about 85%, at least about 90%,
at least about 95%, at least about 96%, at least about 97%, at
least about 98%, at least about 99%, at least about 99.5% of all
species/molecules/particles are spatially separated with respect to
other species/molecules/particles during detection.
Detection Methods
[0189] In some embodiments, in the systems/methods in which the
species to be detected are partitioned across a plurality of
reaction vessels, the reaction vessels may be interrogated using a
variety of techniques, including techniques known to those of
ordinary skill in the art.
[0190] In a specific embodiment of the present invention, reaction
vessels are optically interrogated. The reaction vessels exhibiting
changes in their optical signature may be identified by a
conventional optical train and optical detection system. Depending
on the detected species (e.g., labeling agent molecules,
dissociated species, particles, etc.) and the operative
wavelengths, optical filters designed for a particular wavelength
may be employed for optical interrogation of the reaction vessels.
In one embodiment, the plurality of reaction vessels of the present
invention is formed directly as part of a fiber optic bundle.
[0191] According to one embodiment, the array of reaction vessels
of the present invention can be used in conjunction with an optical
detection system such as the system described in U.S. Publication
No. 20030027126. For example, according to one embodiment, the
array of reaction vessels of the present invention is formed in one
end of a fiber optic assembly comprising a fiber optic bundle
constructed of clad fibers so that light does not mix between
fibers.
[0192] FIG. 17A shows a non-limiting example of a system of the
present invention according to some embodiments. The system
comprises a light source 252, excitation filter 454, dichromatic
mirror 458, emission filter 460, objective 470, and second
substrate 472. Light 453 given off from light source 452 is passed
through excitation filter 454. The light reflects off dichromatic
mirror 458, passes through objective 470 and shines on second
substrate 472. In some cases, stray light 464 may be reduced by a
stray light reducing function 468, such as an iris or aperture.
Light 471 emitted from the second substrate passes through
objective 472 and emission filter 460 and is observed. The system
may comprise additional components (e.g., additional filters,
mirrors, magnification devices, etc.), as needed for particular
applications, as would be understood by those of ordinary skill in
the art.
[0193] The optical detection system of U.S. Publication No.
20030027126 operates as follows. Light returning from the distal
end of the fiber optic bundle is passed by the attachment to a
magnification changer which enables adjustment of the image size of
the fiber's proximal or distal end. Light passing through the
magnification changer is then shuttered and filtered by a second
wheel. The light then is imaged on a charge coupled device (CCD)
camera. A computer executes imaging processing software to process
the information from the CCD camera and also optionally controls
the first and second shutter and filter wheels. As depicted in U.S.
Publication No. 20030027126, the proximal end of the bundle is
received by a z-translation stage and x-y micropositioner.
[0194] For example, FIG. 18 shows a schematic block diagram of a
system employing a fiber optic assembly 400 with an optical
detection system. The fiber optic assembly 400 comprises a fiber
optic bundle or array 402 that is constructed from clad fibers so
that light does not mix between fibers. An array or system, 400 is
attached to the bundle's distal end 412, with the proximal end 414
being received by a z-translation stage 416 and x-y micropositioner
418. These two components act in concert to properly position the
proximal end 414 of the bundle 402 for a microscope objective lens
420. Light collected by the objective lens 420 is passed to a
reflected light fluorescence attachment with three pointer cube
slider 422. The attachment 422 allows insertion of light from a 75
Waft Xe lamp 424 through the objective lens 420 to be coupled into
the fiber bundle 402. The light from the source 424 is condensed by
condensing lens 426, then filtered and/or shuttered by filter and
shutter wheel 428, and subsequently passes through a ND filter
slide 430. Light returning from the distal end 412 of the bundle
402 is passed by the attachment 422 to a magnification changer 432
which enables adjustment of the image size of the fiber's proximal
or distal end. Light passing through the magnification changer 432
is then shuttered and filtered by a second wheel 434. The light is
then imaged on a charge coupled device (CCD) camera 436. A computer
438 executes imaging processing software to process the information
from the CCD camera 436 and also possibly control the first and
second shutter and filter wheels 428, 434.
[0195] The array of reaction vessels of the present invention may
be integral with or attached to the distal end of the fiber optic
bundle using a variety of compatible processes. In some cases,
microwells are formed at the center of each individual fiber of the
fiber optic bundle and the microwells may or may not be sealed.
Each optical fiber of the fiber optic bundle may convey light from
the single microwell formed at the center of the fiber's distal
end. This feature enables the interrogation of the optical
signature of individual reaction vessels to identify
reactions/contents in each microwell. Consequently, by imaging the
end of the bundle onto the CCD array, the optical signatures of the
reaction vessels are individually interrogatable and may be
detected substantially simultaneously.
[0196] As discussed above, in some embodiments of the present
invention, the plurality of reaction vessels may be sealed, for
example, through the mating of the second substrate and a sealing
component. In some cases, the sealing of the reaction vessels may
be such that the contents of each reaction vessel cannot escape the
reaction vessel. In some cases, the reaction vessels may be sealed
after the addition of a dissociated species and, optionally, a
precursor labeling agent molecule to facilitate detection of the
dissociated species. For embodiments employing precursor labeling
agent molecules, by sealing the contents in some or each reaction
vessel, a reaction to produce the detectable labeling agent
molecule can proceed within the sealed reaction vessels, thereby
producing a detectable amount of a labeling agent molecule that is
retained in the reaction vessel for detection purposes.
Quantification
[0197] According to some embodiments of the present invention, the
methods, systems, and/or devices may be used to determine the
presence and/or a measure of the concentration of a plurality of
analyte molecules (or particles) in a fluid sample based at least
in part on detecting and/or quantifying at least some of a
plurality of dissociated species (or particles or molecules
released from a substrate on which the analyte molecules/particles
are captured). In some cases, there is a correlation between the
percentage of reaction vessels containing one or more dissociated
species and the quantity/concentration of analyte molecules in the
fluid sample. Thus, the quantification method of the present
invention may allow for a determination of a measure of the
concentration of analyte molecules in a fluid sample based on the
percentage of reaction vessels that contain a dissociated species
released from a substrate. In some cases, the concentration of the
analyte molecules in a fluid sample will be linearly proportional
to the quantification of the dissociated species. In other cases,
the measure of concentration of the analyte molecules in a fluid
sample will be related to the determination and/or quantification
of the dissociated species by a non-linear relationship. In some
embodiments, the measure of the concentration of analyte molecules
in a fluid sample will be determined using a calibration curve.
Methods to determine a measure of the concentration of analyte
molecules in a fluid sample are discussed more below.
[0198] Certain embodiments of present invention are distinguished
by the ability to detect and/or quantify low numbers/concentrations
of dissociated species and may be well suited to determine a
measure of the concentration of analyte molecules in a fluid sample
containing very low concentrations of the analyte. It is currently
believed that this ability may be achieved by spatially isolating
individual or small numbers of dissociated species, for example, as
partitioned across an array of reaction vessels, and then detecting
their presence in the reaction vessels. The presence of a
dissociated species in a reaction vessel can be counted in a binary
fashion (e.g., zero when a dissociated species is absent; one when
a dissociated species is present), for example by determining the
presence of a detectable molecule or particle in a reaction vessel
that contains at least one dissociated species.
[0199] In some embodiments, the plurality of dissociated species
may be partitioned such that at least some of the reaction vessels
contain no dissociates species and at least some of reaction
vessels contain at least one or, in certain cases, only one
dissociates species. For example, in some cases, the plurality of
dissociates species may be partitioned such that a statistically
significant fraction of the reaction vessels contain no dissociates
species and a statistically significant fraction of reaction
vessels contain at least one dissociates species. In other cases,
the plurality of dissociated species may be partitioned such that a
statistically significant fraction of the reaction vessels contain
no dissociated species and a statistically significant fraction of
reaction vessels contain only one dissociated species. In either
case, the number of the plurality of reaction vessels and/or
fraction of the plurality of reaction vessels that contain or do
not contain a dissociated species may be determined. The number
and/or fraction of the plurality of reaction vessels that contain a
dissociated species can be related to the concentration of analyte
molecules or particles in the sample. In some embodiments, a
measure of the concentration of analyte molecules or particles in
the fluid sample is determined based on the determination of the
number and/or fraction of the plurality of reaction vessels that
contain a dissociated species. In certain such embodiments, the
measure of the concentration of the analyte molecules or particles
in the fluid sample is determined at least in part by comparison of
a measured parameter to a calibration standard and/or by a Poisson
and/or Gaussian distribution analysis of the number or fraction of
the plurality of reaction vessels that contain a dissociated
species, as discussed more below.
[0200] A "statistically significant fraction" of the reaction
vessels that contain a specified quantity of dissociated species is
defined as the minimum number of reaction vessels that can be
reproducibly determined to contain a dissociates species with a
particular system of detection (i.e., substantially similar results
are obtained for multiple essentially identical fluid samples
comprising the dissociates species) and that is above the
background noise (e.g., non-specific binding) that is determined
when carrying out the assay with a sample that does not contain any
analyte molecules or particles, divided by the total number of
reaction vessels. The statistically significant fraction may be
experimentally determined for a certain assay type and equipment
set up (e.g., for each dissociates species determined, each binding
ligand, etc). In certain embodiments, the percentage of reaction
vessels (e.g., the statistically significant fraction) which
comprises only one or at least one dissociates species is less than
about 10%, less than about 5%, less than about 1%, less than about
0.5%, or less than about 0.1% of the total reaction vessels. In
some cases, the percentage of reaction vessels which do not contain
an dissociates species is at least about 20%, at least about 40%,
at least about 50%, at least about 60%, at least about 70%, at
least about 75%, at least about 80%, at least about 90%, or at
least about 95%, at least about 99%, at least about 99.5%, at least
about 99.9%, or greater, of the total number of reaction
vessels.
[0201] In some embodiments, a measure of the concentration of
analyte molecules or particles in the fluid sample may be
determined at least in part by comparison of a measured parameter
to a calibration standard. For example, the fraction of reaction
vessels that comprise a dissociated species may be calibrated
against a calibration curve to determine a measure of the
concentration of the analyte molecule in the fluid sample. The
calibration curve may be produced by completing the assay with a
plurality of standardized samples of known concentration under the
conditions used to analyze the test samples. A reading may be taken
for the signal related to the detection/quantification of the
dissociated species for each standardized sample, therefore
allowing for the formation of a calibration curve relating the
detection of the dissociated species with a known concentration of
the analyte molecule. The assay may then be completed on a sample
comprising the analyte molecule in an unknown concentration, and
the detection of the dissociated species from this assay may be
plotted on the calibration curve, therefore determining a measure
of the concentration of the analyte molecule in the fluid
sample.
[0202] In one exemplary calibration, four standardized fluid
samples comprising an analyte molecule in varying concentration (w,
x, y, and z) are provided. An assay (e.g., immobilizing the analyte
molecules, dissociating a plurality of dissociated species,
detecting at least a portion of the dissociated species) is carried
out for each sample, and a value corresponding to the detection of
the dissociated species (b, c, d, and e) may be determined. A plot
is produced of the values related to detection of the dissociated
species (b, c, d, and e) versus the concentration of the
standardized samples (w, x, y, and z), as depicted in FIG. 19. The
assay may be then be carried out under substantially identical
conditions on a fluid sample comprising an analyte molecule of
unknown concentration t, wherein the resulting value related to
detection of the dissociated species is f. This value may be
plotted on the graph and a measure of the unknown concentration of
the target analyte in the fluid sample may be interpolated from the
values of the standardized samples. In some cases, the calibration
curve may have a limit of detection, wherein the limit of detection
is the lowest concentration of analyte molecules in a fluid sample
that may be accurately determined. In some cases, the r.sup.2 value
of the calibration curve may be greater than about 0.5, greater
than about 0.75, greater than about 0.8, greater than about 0.9,
greater than about 0.95, greater than about 0.97, greater than
about 0.98, greater than about 0.99, greater than about 0.9999, or
about 1.
[0203] In some embodiments, the concentration of analyte molecules
in the fluid sample may be determined by comparison to a
calibration curve using a system comprising a computer. The
computer may comprise software that may use the data collected to
produce the calibration curve and/or a reading of the measured
concentration of the analyte molecules in the fluid sample. For
example, a fluorescence image of an array comprising the
dissociated species partitioned across the array (e.g., second
substrate) may be collected and analyzed using image analysis
software (e.g., IP Lab, BD Biosciences). The analysis software may
automatically calculate the number of reaction vessels that have
fluorescence intensity over the background intensity and produce a
number indicative of the total number of reaction vessel which
comprises a fluorescent intensity over the background intensity
(e.g., a number that correlates to the number of reaction vessels
which comprise a dissociated species). The number of reaction
vessels which comprise fluorescence intensity over the background
intensity may be divided by the total number of reaction vessels to
give a number correlating to the fraction of reaction vessels which
comprise a dissociated species. The active well fraction may be
compared to a calibration curve to determine a measure of the
concentration of analyte molecules in the fluid sample.
[0204] In some cases, the number of dissociated species that are
detected may or may not be approximately equal to the number of
analyte molecules in the fluid sample. For example, the ratio of
analyte molecules to dissociated species that are detected may be
about 1:1, about 2:1, about 5:1, about 10:1, about 100:1, about
1000:1, about 10000:1, or the like. In some cases, the ratio is
greater than about 1:1, greater than about 10:1, great than about
100:1, greater than about 1000:1, greater than about 10000:1, or
the like. Additionally, the number of dissociated species that are
detected may or may not be approximately equal to the total number
of dissociated species released from the substrate. For example,
the ratio of dissociated species dissociated to dissociated species
that are detected may be about 1:1, about 2:1, about 5:1, about
10:1, about 100:1, about 1000:1, about 10000:1, or the like. In
some cases, the ratio is greater than about 1:1, greater than about
10:1, great than about 100:1, greater than about 1000:1, greater
than about 10000:1, or the like. As another example, the number of
molecules or particles detected on or within the second substrate
is less than about one half, less than about one quarter, less than
about one tenth, less than about one hundredth, less than about one
thousandth, less than about one ten-thousandth, less than about one
hundred-thousandth of the number of the plurality of molecules or
particles released from the first substrate.
[0205] In some embodiments, the concentration of analyte molecules
or particles in the fluid sample that may be substantially
accurately determined is less than about 5000 fM, less than about
3000 fM, less than about 2000 fM, less than about 1000 fM, less
than about 500 fM, less than about 300 fM, less than about 200 fM,
less than about 100 fM, less than about 50 fM, less than about 25
fM, less than about 10 fM, less than about 5 fM, less than about 2
fM, less than about 1 fM, less than about 0.5 fM, less than about
0.1 fM, or the like. In some embodiments, the concentration of
analyte molecules or particles in the fluid sample that may be
substantially accurately determined is between about 5000 fM and
about 0.1 fM, between about 3000 fM and about 0.1 fM, between about
1000 fM and about 0.1 fM, between about 1000 fM and about 1 fM,
between about 100 fM and about 1 fM, between about 100 fM and about
0.1 fM, or the like. The concentration of analyte molecules or
particles in a fluid sample may be considered to be substantially
accurately determined if the measured concentration of the analyte
molecules or particles in the fluid sample is within about 10% of
the actual (e.g., true) concentration of the analyte molecules or
particles in the fluid sample. In certain embodiments, the measured
concentration of the analyte molecules or particles in the fluid
sample may be within about 5%, within about 4%, within about 3%,
within about 2%, within about 1%, within about 0.5%, within about
0.4%, within about 0.3%, within about 0.2% or within about 0.1%, of
the actual concentration of the analyte molecules or particles in
the fluid sample. In some cases, the measure of the concentration
determined differs from the true (e.g., actual) concentration by no
greater than about 20%, no greater than about 15%, no greater than
10%, no greater than 5%, no greater than 4%, no greater than 3%, no
greater than 2%, no greater than 1%, or no greater than 0.5%. The
accuracy of the assay method may be determined, in some
embodiments, by determining the concentration of analyte molecules
in a fluid sample of a known concentration using the selected assay
method.
[0206] Without being limited by theory, the quantification method
is believed to be driven in part by the fact that the number and
volume of reaction vessels employed govern the dynamic range of
concentrations that can be determined. That is, based on the number
and volume of the reaction vessels in an array of the present
invention, an estimate can be made of the range of concentrations
of detected molecules in the solution partitioned across the
vessels that allows for a measure of the concentration to be
determined using certain methods of the present invention.
[0207] For example, for an array comprising approximately
2.4.times.10.sup.5 reaction vessels, each having a volume of
approximately 50 fL, a solution having a concentration of
approximately 4.times.10.sup.-11M dissociated species in a fluid
sample will yield, on average, one dissociated species per reaction
vessel. However, it is important to note that distributing a fluid
sample having a dissociated species concentration within the
appropriate range into an array of reaction vessels will not result
in the distribution of exactly one dissociated species per each
reaction vessel; statistically, some vessels will have multiple
dissociated species while others will have no dissociated species.
In the case where the ratio of vessels containing one or more
dissociated species to the number of vessels containing no
dissociated species is high, the data may be fit to a Gaussian
distribution. As the ratio of reaction vessels containing a
dissociated species to the number of vessels containing no
dissociated species approaches zero, the Poisson distribution may
be applied to the data. This limiting distribution may be used to
calculate the probability of rare events occurring in a large
number of trials. For example, based on Poisson statistics, for a
concentration of approximately 4.times.10.sup.-11 M, a distribution
between zero and five dissociated species per container is
predicted, with the most probable values being zero and one.
[0208] Equation 1 can be used to determine the probability of
observing events based on the expected average number of events per
trial, .mu.:
P.sub..mu.(v)=e.sup.-.mu.(.mu..sup.v/v!) Equation 1:
where v is the number of events observed (e.g., the number of
reaction vessels) and .mu. is the expected average number of events
per trial (e.g., the average number of dissociated species per
reaction vessel.
[0209] If the concentrations used are much less than approximately
4.times.10.sup.-11 M, the expected average number of dissociated
species per well becomes exceptionally low, the distribution is
narrowed, and the probability of observing anything other than zero
or one dissociated species per well is improbable in all
experimental cases. At these low concentrations, the relationship
between the percentage of active reaction vessels and the bulk
dissociated species concentration is approximately linear. Thus,
based on this knowledge, the array of the present invention can be
used to determine the concentration of a dissociated species by a
simple digital readout system (e.g., "counting" of active wells) as
described herein in combination with a suitable calibration.
[0210] According to one embodiment, the quantification method of
the present invention can be performed as follows. The method
employs a digital readout system (also referred to as a "binary
readout system") that involves first detecting the dissociated
species in the plurality of reaction vessels by any detection
method as described herein. The number of reaction vessels which
comprise a dissociated species is then counted and a percentage of
the total number of reaction vessels which comprise a dissociate
species is calculated. That is, utilization of a yes or no
response, in conjunction with the high-density array of reaction
vessels, permits the digital readout of bulk concentrations of
dissociated species. In some embodiments, this readout is
accomplished by counting the number of reaction vessels containing
at least one labeling agent molecule, with the resulting number of
reaction vessels comprising a labeling agent molecule corresponding
to the number of reaction vessels comprising a dissociated species.
Given the large number of reaction vessels simultaneously
interrogated in the array of the present invention, the ratio of
dissociated species to reaction vessels may be at least about
1:100, at least about 1:1000, at least about 1:10,000, as the large
number of reaction vessels provides a statistically significant
signal even at this low ratio.
[0211] In some embodiments, without being limited by theory, it is
believed that the quantification method of the present invention
may only be limited by the number of individual reaction vessels
that can be fabricated and interrogated. Thus, expanding the number
of reaction vessels may increase both the dynamic range and the
sensitivity of the assay. For example, increasing the number of
reaction vessels by a factor of ten may decrease the ratio of
dissociated species to reaction vessels by a factor of ten, thereby
increasing the dynamic range and/or sensitivity of the assay. As
mentioned above, in some embodiments, an array will comprise
between about 1,000 and about 50,000, between about 1,000 and about
1,000,000, between about 1,000 and about 10,000, between about
10,000 and about 100,000, between about 100,000 and about
1,000,000, between about 1,000 and about 100,000, between about
50,000 and about 100,000, between about 20,000 and about 80,000,
between about 30,000 and about 70,000, between about 40,000 and
about 60,000, or about 50,000, or the like, reaction vessels.
[0212] In some embodiments, accuracy of this technique may be
compromised above and below the thresholds of the dynamic range.
For example, as the concentration of the dissociated species goes
below the lower limit of the dynamic range, the number of
dissociated species may be too low to observe a sufficient number
of occupied wells. In such a situation, the number of reaction
vessels could be decreased in order to make sure that at least some
(e.g., a statistically significant number) of them are occupied by
a dissociated species, the volume of the reaction vessels could be
increased, and/or the sample tested could be concentrated. Results
for extremely dilute concentrations may have large relative errors
associated with them, due to the very small number of reaction
vessels that are expected to show activity. In other cases, the
ultimate upper limit to this technique may occur when 100% of the
reaction vessels contain at least one dissociated species. At this
limit, discrimination between two solutions of high dissociated
species concentrations may not be feasible. In such a situation, to
provide a more accurate test, a greater number of reaction vessels
could be used, and/or the volume of each reaction vessel could be
reduced, and/or the concentration of the sample could be reduced,
e.g., through serial dilutions.
[0213] In the range where the fraction of reaction vessels
containing at least one dissociated species is less than about 20%,
the probability that any well contains two or more dissociated
species is very small and the number of dissociated species closely
matches the number of occupied reaction vessels. Between 20%
occupied and 100% occupied, an increasing number of wells may
contain more than one dissociated species, however Gaussian
statistics can still be used to correlate occupancy fraction with
concentration with reasonable accuracy until the occupancy fraction
approaches 100%.
[0214] As alluded to above, the practical dynamic range of the
method may be increased in several ways. In one approach, the
sample and/or solution carrying the dissociated species may be
diluted by a factor of 10 or more. Both the solution comprising the
dissociated species and the diluted solution comprising the
dissociated species may be assayed concurrently using the method of
this invention. The dynamic ranges of the two assays may overlap,
but be offset by the dilution factor, hence extending the dynamic
range.
[0215] In some embodiments, multiple arrays of reaction vessels can
be used, each array having reaction vessels with differing volumes,
differing binding surface areas, or differing density and/or type
of capture components on the binding surface. These configurations
can be constructed as either distinct arrays or as one large array
with distinct sub-arrays with varying characteristics. Since the
probability of a dissociated species being detected in a given
reaction vessel can be related to volume, binding surface area, and
capture component density, the sub-arrays may be designed to
provide different sensitivity ranges. Thus, with such
configurations the effective range of the combined array may be
extended.
[0216] In certain embodiments, after partitioning of the solution
containing dissociated species across an array of reaction vessels,
less than about 20% of the total number of the plurality of
reaction vessels will contain at least one dissociated species
(i.e. at least 80% of reaction vessels will be free of dissociated
species). Under such circumstances, the number of reaction vessels
containing at least one dissociated species will typically fall
within the linear range of a Poisson distribution. In another
embodiment, more than about 20% but less than about 60% of the
total number of reaction vessels contains at least one dissociated
species. Under such circumstances, the number of reaction vessels
containing at least one dissociated species will typically fall
within the non-linear range of a Poisson distribution. In another
embodiment, more than about 60% but less than about 95% of the
total number of reaction vessels contains at least one dissociated
species. Under such circumstances, the number of reaction vessels
containing at least one dissociated species will typically fall
within the highly non-linear range of a Poisson distribution. In
embodiments where greater than about 60% of the total number of
reaction vessel contains at least one dissociated species, it may
be desirable to decrease the percentage of reaction vessels which
comprise at least one dissociated species (e.g., to less than about
20%). This may be accomplished using any suitable technique
discussed herein, for example, diluting the fluid comprising the
dissociated species and/or increasing the number of reaction
vessels. In some cases, less than about 1%, less than about 5%,
less than about 10%, less than about 20%, less than about 40%, less
than about 60%, less than about 80%, less than about 90%, less than
about 95%, or less than about 99% of the total number of the
plurality of reaction vessels will contain at least one dissociated
species. In certain embodiments, more than about 1%, more than
about 5%, more than about 10%, more than about 20%, more than about
40%, more than about 60%, more than about 80%, more than about 90%,
more than about 95%, or more than about 99% of the total number of
the plurality of reaction vessels will contain no dissociated
species.
[0217] In some embodiments, the invention provides a method of
determining the concentration of the dissociated species in a
fluid, the method comprising dividing the fluid containing the
dissociated species into a plurality of second, smaller fluid
samples of essentially equal volume so that at least some (e.g., a
statistically significant fraction) of the second, smaller fluid
samples contain either no dissociated species or a single
dissociated species; determining the presence or absence of a
dissociated species in each of the second, smaller fluid samples so
as to identify the number of second, smaller fluid samples that
contain a dissociated species; and determining the concentration of
dissociated species in the fluid sample to be tested from the
number of second, smaller samples that contain the dissociated
species.
[0218] In certain embodiments, the present invention provides a
method for determining the concentration of dissociated species in
a fluid, the method comprising the partitioning at least a portion
of the dissociated species in the fluid across a plurality of
reaction vessels so that a statistically significant fraction of
the reaction vessels contain a dissociated species and a
statistically significant fraction of the reaction vessels contain
no dissociated species; determining the presence or absence of a
dissociated species in each reaction vessel to identify the number
of reaction vessels that contain a dissociated species and/or to
identify the number of reaction vessels that contain no dissociated
species; and determining the concentration of dissociated species
in the fluid at least in part from the number of reaction vessels
that do or do not contain a dissociated species. In some
embodiments, at least about 99%, at least about 95%, at least about
90%, at least about 80%, at least about 40%, at least about 20%, at
least about 10% at least about 5%, at least about 1%, and the like
of the reaction vessels do not contain a dissociated species. In
some embodiments, the concentration of dissociated species in the
fluid sample is determined at least in part using a calibration
curve, a Poisson distribution analysis and/or a Gaussian
distribution of the number of reaction vessels that contain at
least one or one dissociates species. In other embodiments, the
concentration of dissociated species in the fluid sample is
determined at least in part by a Gaussian distribution analysis of
the number of reaction vessels that contain a dissociated
species.
[0219] In certain embodiments, the present invention provides a
method of determining the concentration of dissociated species in a
fluid, the method comprising exposing the fluid to a plurality of
reaction vessels under conditions so that at least one dissociated
species is captured in at least some of the reaction vessels,
wherein each reaction vessel comprises a microwell and an optional
sealing component and each reaction vessel defines a binding
surface that has a capture component immobilized thereon;
determining the presence or absence of a dissociated species in
each reaction vessel so as to identify the number of reaction
vessels that contain a dissociated species and/or the number of
reaction vessels that do not contain a dissociated species; and
determining the concentration of dissociated species in the fluid
sample to be tested from the number of reaction vessels that
contain and/or do not contain a dissociated species. In some
embodiments, at least about 99%, at least about 95%, at least about
90%, at least about 80%, at least about 50%, at least about 20%, at
least about 10% at least about 5%, at least about 1%, and the like
of the reaction vessels contain either zero or one dissociated
species. In some embodiments, the concentration of dissociated
species in the fluid sample is determined at least in part using a
calibration curve, a Poisson distribution analysis and/or a
Gaussian distribution analysis of the number of reaction vessels
that contain at least one or one dissociated species.
[0220] In certain embodiments, the present invention provides a
method of determining the concentration of dissociated species in a
fluid sample to be tested, the method comprising partitioning at
least a portion of the dissociated species in the fluid into a
plurality of reaction vessels, so that, for substantially all of
the reaction vessels, each reaction vessel contains either no
dissociated species or a single dissociated species; determining
the presence or absence of a dissociated species in a plurality of
reaction vessels to provide a fraction of the interrogated reaction
vessels that contain a dissociated species; and determining the
concentration of dissociated species in the fluid from the fraction
of interrogated reaction vessels that contain a dissociated
species.
[0221] In certain embodiments, the present invention provides a
method of determining the concentration of dissociated species in a
fluid, the method comprising partitioning the fluid into a
plurality of second, smaller fluid samples of equal volume so that
at least some of the second, smaller fluid samples contain either a
single dissociated species or no dissociated species, (b)
determining the presence or absence of a dissociated species in at
least a subset of the second samples so as to identify the fraction
of second samples in the subset that contain a dissociated species;
and determining the concentration of dissociated species in the
sample to be tested from the fraction of second samples of the
subset that contain the dissociated species.
[0222] In some embodiments, a method of the present invention may
be used for the detection of dissociated species in a fluid. For
example, a method of detecting dissociated species in a fluid may
comprise providing a fluid containing the dissociated species and
an array, the array comprising a plurality of reaction vessels;
contacting the array with the fluid such that the ratio of the
number of dissociated species in the fluid contacted with the array
to the number of reaction vessels in the array is less than 1:1;
and determining the number of reaction vessels which contain a
dissociated species. In some cases, the ratio of the number of
dissociated species in the fluid contacted with the array to the
number of reaction vessels in the array is less than about 1:5,
less than about 1:10, less than about 1:100, or less than about
1:500.
[0223] In certain embodiments, a method of detecting dissociated
species in a fluid according to the invention comprises providing a
fluid and an array, the fluid comprising at least one dissociated
species at a first concentration, the array comprising a plurality
of reaction vessels; diluting the fluid to create a diluted fluid,
wherein the diluted fluid comprises the dissociated species at a
second concentration,; contacting the array with the diluted fluid
such that the ratio of dissociated species to the total number of
reaction vessels in the array is between 1:1 and 1:500; and
determining the number of vessels of said array which contain a
dissociated species. In some cases, the ratio is less than about
1:1, less than about 1:5, less than about 1:10, less than about
1:100, or less than about 1:500.
[0224] The following examples are included to demonstrate various
features of the invention. Those of ordinary skill in the art
should, in light of the present disclosure, will appreciate that
many changes can be made in the specific embodiments which are
disclosed while still obtaining a like or similar result without
departing from the scope of the invention as defined by the
appended claims. Accordingly, the following examples are intended
only to illustrate certain features of the present invention, but
do not necessarily exemplify the full scope of the invention.
Example 1
[0225] This example outlines the materials used in the following
examples. Optical fiber bundles were purchased from Schott North
America, Inc (Southbridge, Mass.). In this example, the core glass
comprised barium, lanthanum, boron, silica, and aluminum. The
refractive index of the core was 1.694, and the density was 4.23
g/cc. The cladding glass comprised silica, lead, potassium, sodium,
and aluminum. The refractive index of the clading was 1.559, and
the density was 3.04 g/cc. The fiber array was a bundle of 50,000
individual fibers, each with a core diameter of 4.5 .mu.m and the
center-to-center spacing of the cores was 8 um.
[0226] Non-reinforced gloss silicone sheeting was obtained from
Specialty Manufacturing Inc. (Saginaw, Mich.). Hydrochloric acid,
3-aminopropyl trimethoxysilane, anhydrous ethanol, molecular
biology grade Tween.RTM.20, and N,N-Dimethyl formamide were all
obtained from Sigma-Aldrich (Saint Louis, Mo.). Phosphate buffered
saline, Blocker.TM. BSA (10%), Zeba Desalt spin columns,
NHS-ss-biotin and NHS-LC-biotin were obtained from Pierce
(Rockford, Ill.). Resorufin-beta-D-galactopyranoside,
streptavidin-beta-galactosidase, and 1 um functionalized magnetic
microspheres were purchased from Invitrogen (Carlsbad, Calif.).
Monoclonal anti-human TNF-alpha capture antibody, polyclonal
anti-human TNF-alpha detection antibody, and recombinant human
TNF-alpha were purchased from R&D systems, Inc (Minneapolis,
Minn.). The fiber polisher and polishing consumables were purchased
from Allied High Tech Products, Inc. (Rancho Dominguez,
Calif.).
Example 2
[0227] The example outlines the preparation of 1 um magnetic bead
functionalized with TNF-alpha capture antibody. 20 uL of
amine-reactive bead stock was washed three times in 0.1M sodium
borate coating buffer at pH 9.5 using a microtube magnetic
separation device (BioMag.RTM. Tube Separator; Polysciences Inc.,
Warrington, Pa.). 500 ug TNF-alpha capture antibody was dissolved
in 183.5 uL of sodium borate coating buffer. 104 uL of 3M ammonium
sulfate was added to the antibody solution. 135 uL of the resulting
antibody solution was added to the cleaned 20 uL bead aliquot and
mixed at 37.degree. C. for 24 hours. After incubation, the
supernatant was removed using the magnetic separator, and 200 uL of
PBS buffer containing 0.5% BSA and 0.05% Tween.RTM.20 was added to
the beads. The beads were blocked overnight (.about.8 hours) at
37.degree. C. The functionalized and blocked beads were washed 3
times with 1 mL PBS buffer containing 0.1% BSA and 0.05%
Tween.RTM.20. The beads were diluted to 1.5 mL in PBS containing
0.1% BSA, 0.5% Tween.RTM.20, and 0.02% sodium azide. 100 uL
aliquots were stored at 4.degree. C. for later use.
Example 3
[0228] The following is an example of the preparation of TNF-alpha
detection antibody (binding ligand) comprising a disulfide
cleavable biotin linkage. 100 ug of TNF-alpha detection antibody
(R&D Systems, AF-2,0-NA) was dissolved in 100 uL PBS pH 7.4
buffer. 164 uL of PBS pH 7.4 buffer was added to a tared vial
containing 1 mg sulfo-NHS-ss-biotin, yielding a 10 mM stock
solution. 1.34 uL of the 10 mM stock solution was added to the 100
uL antibody solution, equating to a 20-fold molar excess of the
biotin label. The antibody/NHS-ss-biotin solution was mixed for 1
hr at room temperature. The antibody-ss-biotin conjugate was
purified using a Zeba Desalt Spin Column. The purification was
performed according to the Pierce protocol for product #89882. The
purified conjugate was diluted up to 2 mL, making a 50 ug/mL stock
solution. 50 uL aliquots of this 50 ug/mL solution were stored at
-20.degree. C. for later use.
Example 4
[0229] The following is an example of the capture of TNF-alpha on
magnetic beads and formation of immunocomplexes. Solutions
containing TNF-alpha were incubated with suspension of beads
functionalized with capture antibody, as described in Example 2,
for 1 h at 37.degree. C. The beads were separated and washed three
times in PBS. The beads were resuspended and incubated with 2 nM of
detection antibody (binding ligand), prepared as described in
Example 3, for 30 min at 37.degree. C. The beads were separated and
washed three times in PBS. The beads were then incubated with 200
pM of streptavidin-beta-galactosidase (converting agent) for 30
minutes at 37.degree. C., separated, and washed three times in
PBS.
Example 5
[0230] The following is an example of the cleaving of disulfide
linkages in the TNF-alpha magnetic beads containing immunocomplex
as prepared in Example 4. Magnetic beads containing the complete
immunocomplex were washed three times in 100 uL of 5.times.PBS
containing 0.1% Tween.RTM.20 for 2 minutes each time. The final
wash volume was removed and 100 uL of 10 mM beta-mercaptoethanol
containing 10 mM EDTA in PBS buffered solution was added to the
beads. The beads were resuspended and incubated at 37.degree. C.
for 60 minutes while shaking (Jitterbug Model 130000; Boekel
Scientific, Feasterville, Pa.) at 500 RPM. The entire liquid volume
containing dissociated species was separated from the beads and
stored at 4.degree. C.
Example 6
[0231] The following is an example of the cleaving of a
photo-cleavable linkage in a TNF-alpha magnetic bead containing an
immobilized immunocomplex. A 96 well white Nunc Maxisorb plate on
which were immobilized complete immunocomplexes, prepared similarly
as described in the above Examples, except comprising
photo-cleavable biotin linkages as opposed to disulfide cleavable
biotin linkage on the detection antibody (binding ligand) were
washed three times in 150 uL of 5.times.PBS containing 0.1%
Tween.RTM.20 for 2 minutes with 500 RPM mixing on a VWR plate
shaker each time. The final wash volume was removed and 50 uL of
1.times.PBS solution was added to the wells. The plate was placed
.about.5 cm from the UV light source (365 nm black light bulbs, 15
watt) for 30 to 60 minutes to activate the UV photo-cleavage of the
biotin linkage. Photo-cleaved samples were transferred to a fresh
plate following separation from the Nunc plate.
Example 7
[0232] The following is an example of the preparation of
biotinylated array for capture of released reporter molecules
(dissociated species). Optical fiber bundles approximately 5 cm
long were polished on a polishing machine using 30, 9, and 1
micron-sized diamond lapping films. The polished fiber bundles were
chemically etched in a 0.025 M HCl solution for 115 seconds, and
then immediately submerged into water to quench the reaction. To
remove impurities from etching, the etched fibers were sonicated
for 5 seconds in water and washed in water for 5 min. The fibers
were then dried under vacuum and exposed to air plasma for 5
minutes to clean and activate the glass surface. The arrays were
silanized for 30 minutes in a 2% silanization solution that was
prepared by mixing 950 uL of anhydrous ethanol, 50 uL of water, and
20 uL of 3-aminopropyl trimethoxysilane, and then washed with
anhydrous ethanol for 10 min and dried under nitrogen. The amine
silanized arrays were then biotinylated using the following
procedure.
[0233] 50 mg NHS-LC-biotin was dissolved in 1.1 mL anhydrous DMF to
make a 100 mM stock solution. 1 mL of the 100 mM stock solution was
diluted with 4 mL PBS buffer pH 7.4 to make 5 mL of a 20 mM working
solution. The working solution was distributed in 500 uL aliquots
into multiple 20 ml scintillation vials. Each vial can accommodate
up to 8 arrays. The arrays were placed into the biotin solution and
incubated with mixing for 60 minutes. The arrays were removed from
the biotin solution and rinsed briefly with PBS buffer, followed by
washing two times in 15 mL PBS buffer for 10 minutes. The
biotinylated arrays were used immediately, or were stored for up to
2 days in PBS buffer for later use.
Example 8
[0234] The following is an example of the capture of
streptavidin-beta-galactosidase reporter (dissociated species)
released from the magnetic beads, as described above in Example 5,
on functionalized fiber arrays prepared as described as described
in Example 7. 100 uL cleaved samples were received in microtiter
plates in a 10 mM beta-mercaptoethanol/PBS buffered solution. The
100 uL samples were transferred to 1.5 mL reaction tubes and
capped. A small hole was made in the tube top, and a biotin
functionalized fiber array was inserted into and suspended in the
100 uL sample. The arrays were incubated in the cleaved samples for
60 minutes at room temperature with 250 RPM mixing on a benchtop
vortexer. Arrays were transferred to a second microtube containing
PBS buffer, which was used to store the array for subsequent
examination on the imaging system.
Example 9
[0235] The following is an example of instrumental set-up that may
be employed to analyze the fiber optic arrays prepared as described
in Example 8. An upright epifluorescence microscope containing a
mercury light source, filter cubes, objectives, and a CCD camera
was used for acquiring fluorescence images. The set-up is similar
to that as described above in FIG. 17A and is also pictured in FIG.
17B. The imaging system used for collecting the data integrated an
Olympus BX-61 microscope system with a Cooke Corporation Sensicam
QE CCD camera, 474. The BX-61 microscope is an upright microscope
specifically designed for fluorescence detection. The scope housed
UIS2 optics, which, in comparison to standard optics, offers
increased signal-to-noise ratio, high light transmission, and
diverse illumination capabilities. 10.times. and 20.times. UMPlanFl
objectives (e.g., 470), which have a NA of 0.3 and 0.5,
respectively, were installed on the BX-61 microscope. Wavelength
specific filter sets are held in a cube, which is stored in a
rotating wheel within the microscope. The BX-61 can hold 6 cubes
for easy access to wavelengths specific for a number of
fluorophores. Filter sets were purchased from Chroma Technology
Corporation and are compatible with the cubes on the BX-61. The
resorufin filter cube (Chroma Technology filter set #41010) was
used for all of the enzyme amplification experiments described
herein. This filter set uses a HQ577/10x excition filter, a 585
long-pass dichroic mirror, and a HQ620/60m emission filter.
[0236] A 6-axis mechanical platform was constructed beneath the
microscope stage, which was used to house a non-reinforced silicone
gasket material (sealing component) to be applied to the fiber
optic array. Arrays were mounted on the microscope stage using a
fiber array holder fabricated to secure and position the fiber
array on the stage. A droplet of beta-galactosidase substrate (RDG)
(precursor labeling agent molecule) was placed on the silicone
gasket material, and put into contact with the distal end of the
fiber array. The mechanical platform was used to move the silicone
sheet into contact with the distal end of the etched optical fiber
array, creating an array of isolated femtoliter reaction vessels.
The number of wells that bound an enzyme molecule carried by the
dissociated species correlated to the bulk dissociated species
concentration, which, in turn, correlated with the concentration of
TNF analyte in the initial fluid sample applied to the magnetic
bead substrate.
Example 10
[0237] The following example outlines a standard procedure for a
complete assay according to one embodiment of the present
invention. The following procedure is a basic protocol that may be
completed for any suitable sample comprising a plurality of analyte
molecules, and the assay components (e.g., binding ligands, capture
components, etc.) may be selected according to the methods
described herein. This protocol or portions of it was followed when
performing the assays discussed in Examples 11-14.
Chemical and Materials:
[0238] The following solutions should be prepared:
[0239] 1. 1.times.PBS, pH 7.5: The solution should be 0.2 um
filtered and may be diluted from 10.times.PBS (Omnipure cat#6506).
To prepare, mix 100 mL 10.times.PBS and 900 mL Milli-Q water to
form 1 liter total. The solution may be stored at room
temperature.
[0240] 2. Wash Buffer: The solution comprises 5.times.PBS+0.1%
Tween.RTM.20 and was 0.2 um filtered. To prepare, mix 250 uL 100%
Tween.RTM.20 and 125 mL Ultrapure Water and 125 mL 10.times.PBS to
form 250 mL total. The solution may be stored at room temperature
for up to one week
[0241] 3. Detection Ab Buffer: The solution comprises 10% BSA+0.1%
Tween.RTM.20. To prepare mix 5 g BSA, 45 mL 1.times.PBS and 50 uL
100% Tween.RTM.20 to form 50 mL total. The solution may be stored
at 4.degree. C. for up to one week
[0242] 4. Dilution Buffer: The solution comprises 0.5% BSA+0.1%
Tween.RTM.20. To prepare mix 2.5 mL 10% BSA, 47.5 mL 1.times.PBS,
47.5 uL 100% Tween.RTM.20 to form 50 mL total. The solution should
be discarded after completing the assay.
[0243] 5. Reporter Buffer: The solution comprises 1.times.PBS+1 mM
MgCl.sub.2. To prepare, mix 50 uL 1M MgCl.sub.2, 50 mL 1.times.PBS
to form 50 mL total. The solution should be stored at room
temperature.
Preparation of the 96 Well Plate:
[0244] 1. Obtain coupled beads (e.g., MyOne Beads (Invitrogen)
prebound with aTNF Ab BAF210 (R&D Systems)). For example, the
beads may be at 40 ug Ab/mg Dynabeads. Briefly vortex beads at
approximately setting 8 until resuspended. Pipette up and down if
necessary. Sonicate for approximately 10 seconds. Dilute beads to
approximately 0.6 ng Ab/uL (vortex and sonicate all dilutions
before use or subsequent dilution) in dilution buffer. Add 100 uL
beads to appropriate wells of a 96-well plate. Add 100 uL of
dilution buffer only to control wells.
[0245] 2. Bead Preparation: Place plate on bar magnet and wait 30
seconds. Pour Dilution Buffer into a reagent reservoir. Using
multichannel, remove all liquid from each well. Remove plate from
bar magnet. Using multichannel, add 100 uL of Dilution buffer to
each well. Pipette up and down vigorously 15 times to resuspend
beads. Cover plate with hard plastic lid. Incubate on plate shaker
at 500 RPM for 2 minutes. Repeat wash 2.times. more for a total of
3 washes.
Capture TNF-Alpha on Plate:
[0246] 1. Dilute TNF-alpha (R&D systems 210-TA/CF) to
appropriate concentration(s) in dilution buffer
[0247] 2. Capture antigen: Aliquot 100 uL/well of TNF-alpha
solution or dilution buffer only (for NSB wells) with single
channel pipette. Cover plate with hard plastic lid. Incubate on
plate shaker at 500 RPM for 1 hour. After 1 hour, beads will have
partially settled. Pipette up and down vigorously 15 times to
resuspend beads. Continue to incubate for 1 additional hour.
[0248] 3. Wash Plate: Place plate on bar magnet and wait 30
seconds. Pour Wash Buffer into a reagent reservoir. Using
multichannel remove all liquid from each well. Remove plate from
bar magnet. Using multichannel, add 100 uL of wash buffer to each
well. Pipette up and down vigorously 15 times to resuspend beads.
Cover plate with hard plastic lid. Incubate on plate shaker at 500
RPM for 2 minutes. Repeat wash 2.times. more for a total of 3
washes.
Sandwich TNF-Alpha on Beads Using Biotin-Labeled TNF-Alpha
Detection Antibody
[0249] 1. Dilute releasable Biotin-labeled TNF-alpha detection
Antibody (R&D systems BAF210--modified with Sulfosuccinimidyl
2-(biotinamido)-ethyl-1,3-dithiopropionate (ThermoFisher
Scientific)) in dilution buffer to 0.3 ug/mL. In a 15 mL tube add
and vortex to combine: 42 uL TNF-alpha Detection Antibody (50 ug/mL
Stock) 7 mL Detection Ab buffer to form 7 mL total at 0.3 ug/mL
TNF-alpha Antibody (2 nM)
[0250] 2. Sandwich antigen by binding Detection antibody in each
well: Dump TNF-alpha Antibody solution into a reagent reservoir.
Aliquot 100 .mu.L/well with multichannel pipette. Seal Plate with
clear plastic lid. Incubate on plate shaker at 500 RPM for 1
hour.
[0251] 3. Wash Plate: Place plate on bar magnet and wait 30
seconds. Using multichannel remove all liquid from each well.
Remove plate from bar magnet. Using multichannel, add 100 uL of
wash buffer to each well. Pipette up and down vigorously 15 times
to resuspend beads. Cover plate with hard plastic lid. Incubate on
plate shaker at 500 RPM for 2 minutes. Repeat wash 2.times. more
for a total of 3 washes.
Label TNF-Alpha Detection Antibody with Signal Amplification Enzyme
(Streptavidin-.beta.-Galactosidase)
[0252] 1. Dilute Streptavidin labeled TNF-alpha detection
.beta.-galactosidase (SA-.beta.-gal) in dilution buffer to 500 pM:
In a 15 mL tube add and vortex to combine 3.1 uL SA-.beta.-gal (1.6
uM Stock), 10 mL Dilution buffer, to form 10 mL total of 500 .mu.M
SA-.beta.-gal
[0253] 2. Label Detection antibody with signal amplification
reporter SA-.beta.-gal: Dump SA-.beta.-gal solution into a reagent
reservoir. Aliquot 100 uL/well with multichannel pipette
Seal Plate with clear plastic lid. Incubate on plate shaker at 500
RPM for 30 minutes.
[0254] 3. Wash Plate: Place plate on bar magnet and wait 30
seconds. Using multichannel remove all liquid from each well.
Remove plate from bar magnet. Using multichannel, add 100 uL of
wash buffer to each well. Pipette up and down vigorously 15 times
to resuspend beads. Cover plate with hard plastic lid. Incubate on
plate shaker at 500 RPM for 5 minutes. Repeat wash 3.times. more
for a total of 4 washes.
[0255] 4. Transfer Beads: Place plate on bar magnet and wait 30
seconds. Using multichannel remove all liquid from each well.
Obtain a second 96-well plate. Add 75 uL of wash buffer to each
well of the original plate. Transfer entire volume from each well
to the corresponding well of the second plate. Repeat previous
steps to ensure complete bead transfer.
Cleave Detection Ab
[0256] 1. Wash Plate: Place plate on bar magnet and wait 30
seconds. Using multichannel remove all liquid from each well.
Remove plate from bar magnet. Using multichannel, add 100 uL of
wash buffer to each well. Pipette up and down vigorously 15 times
to resuspend beads. Cover plate with hard plastic lid. Incubate on
plate shaker at 500 RPM for 2 minutes. Repeat wash 2.times. more
for a total of 3 washes.
[0257] 2. BME/EDTA Incubation: Dilute Beta Mercaptoethanol (BME) to
10 mM in PBS. Add EDTA to 10 mM final concentration. 0.2 uM-filter
the solution. Using multichannel remove all liquid from each well.
Add 50 uL of BME/EDTA to each well to be cleaved using single
channel pipette. Incubate plate for 60 minutes at 37.degree. C.
Remove entire volume from each well and transfer to corresponding
well in new plate. Discard unused BME/EDTA solution.
Example 11
[0258] The following is an example of the quantification of Tumor
Necrosis Factor alpha (TNF.alpha.) using the protocol of Example
10. Magnetic beads coated in a monoclonal antibody to human TNF
were prepared as described in Example 10. Solutions were then made
containing human TNF at concentrations from 0 fM to 1000 fM spiked
into bovine serum. The magnetic beads and TNF serum solutions were
then combined in a microtiter plate to give final concentrations of
magnetic beads of approximately 1 ug/well. The beads and serum were
mixed at 37.degree. C. for 1 hour. A magnet was then applied to the
plate to separate the magnetic beads and serum, and the serum was
removed. The beads were then subjected to cycles of washing,
exposure to biotinylated detection antibody (first binding ligand),
exposure to streptavidin-beta-galactosidase (second binding
ligand), and magnetic separation as described in Example 10. The
immunocomplex was then released from the beads by adding 100 ul of
10 mM beta-mercaptoethanol to each well of the plate. Solutions
containing the dissociated species were then incubated with biotin
functionalized fiber arrays to capture the released
immunocomplexes. Single dissociated complexes were then detected by
fluorescently imaging single beta-galactosidase molecules in the
wells of the fiber arrays as described above in Examples. These
images were then analyzed and the number of wells containing a
single beta-galactosidase enzyme was determined. This number was
converted to "% active wells" by ratio of the number of active
wells to the total number of wells in the array. FIG. 20A shows a
plot of % active wells against concentration of TNF in the serum
samples. FIG. 20B shows a magnified view of the lower end of the
graph in FIG. 20A. Statistical analysis of these data showed that
the limit of detection for TNF (defined as 3 standard deviations of
the background signal over background) was 0.2 fM, compared to 1250
fM for a standard ELISA using the same reagents. Table 1 shows the
tabulated limit of detection (LOD) for the methods presented in
FIGS. 20-23 as compared to a standard enzyme-linked immunosorbent
assay (ELISA) using essentially similar reagents;
TABLE-US-00001 TABLE 1 Presented Methods ELISA Protein LOD (fM) LOD
(fm) Improvement TNF-alpha 0.2 1250 4000x PSA 2 2000 1000x IL-6 0.2
400 2000x VEGF 0.5 500 1000x
Example 12
[0259] The following is an example of the quantification of
Prostate Specific Antigen (PSA). Magnetic beads coated in a
monoclonal antibody to human PSA were prepared using the method
described above Example 10. Solutions were then made containing
human PSA at concentrations from 0 fM to 200 fM spiked into bovine
serum. The magnetic beads and PSA serum solutions were then
combined in a microtiter plate to give final concentrations of
magnetic beads of 0.7 ug/well. The beads and serum were mixed at
25.degree. C. for 1 hour. The samples were then processed as for
TNF in Example 11 and the % active wells arising from the released
single enzymes was determined. FIG. 21 shows a plot of % active
wells against concentration of PSA in the serum samples. The limit
of detection for PSA is 2 fM, compared to 2000 fM for a standard
ELISA using the same reagents (Table 1).
Example 13
[0260] The following is an example of the quantification of
Interleukin 6 (IL-6) Magnetic beads coated in a monoclonal antibody
to human IL-6 were created using the method described above in
Example 10. Solutions were then made containing human IL-6 at
concentrations from 0 fM to 20 fM spiked into bovine serum. The
magnetic beads and IL-6 serum solutions were then combined in a
microtiter plate to give final concentrations of magnetic beads of
0.5 ug/well. The beads and serum were mixed at 25.degree. C. for 1
hour. The samples were then processed as for TNF in Example 11 and
the % active wells arising from the released single enzymes was
determined FIG. 22 shows a plot of % active wells against
concentration of IL-6 in the serum samples. The limit of detection
for IL-6 is 0.2 fM, compared to 400 fM for a standard ELISA using
the same reagents (Table 1).
Example 14
[0261] The following is an example of the quantification of
Vascular Endothelial Growth Factor (VEGF). Magnetic beads coated in
a monoclonal antibody to human VEGF were created using the method
described above in Example 10. Solutions were then made containing
human VEGF at concentrations from 0 fM to 1000 fM spiked into
bovine serum. The magnetic beads and VEGF serum solutions were then
combined in a microtiter plate to give final concentrations of
magnetic beads of 0.5 ug/well. The beads and serum were mixed at
25.degree. C. for 1 hour. The samples were then processed as for
TNF in Example 11 and the % active wells arising from the released
single enzymes was determined FIG. 23 shows a plot of % active
wells against concentration of VEGF in the serum samples. The limit
of detection for VEGF is 0.5 fM, compared to 500 fM for a standard
ELISA using the same reagents (Table 1).
Example 15
[0262] The following example presents results for several assays
performed using the above described methods.
[0263] FIG. 24 describes the effect of the concentration of
beta-mercaptoethanol on release of dissociated species, in one
embodiment of the invention. The concentration of
beta-mercaptoethanol (bME) used to release SbG was optimized for
signal-to-background to enhance selectivity for specifically bound
molecules. The released SbG was detected on single molecule array
fiber bundles. In this example, 5 mM bME was determined to yield
the best results based on signal-to-background ((ii)/(i)).
[0264] FIG. 25 describes the effect of removing
beta-mercaptoethanol before detection of the dissociated species on
an array. In some cases, beta-mercaptoethanol may adversely affect
the spectrum of the fluorescent product and/or may reduce the
enzymatic activity of SbG. Beta-mercaptoethanol may be removed from
the assay, in some cases, for example by either purifying the
solution of SbG using a filtration column to remove the bME or by
capturing the SbG on fiber arrays and washing the bME away. In this
example, immunocomplexes of TNF-alpha were formed and released by
bME as described in Example 11. The solution containing the
dissociated complexes were (left) passed over a ZEBA column to
remove the bME or (right) were left untreated. These solutions were
then incubated with biotin functionalized fiber arrays (as
described in Example 11) to capture dissociated SbG. The arrays
were then washed in PBS buffer. The graph in FIG. 25 shows that the
enzymatic activity of SbG was maintained with or without removal of
the bME using a ZEBA gel filtration column. The capture and washing
steps were sufficient to remove bME and to allow detection of
single dissociated complexes. In this embodiment, the detection of
SbG was not adversely affected by lack of removal of bME by gel
filtration.
[0265] Non-specific binding (NSB) can be a major limitation on the
sensitivity of an immunoassay. FIG. 26 shows that method of the
present, in this embodiment, can be optimized to result in
non-specific binding of less than about <0.1%. In some cases,
the non-specific binding was reduced to less than about 10 wells in
an 11,000-well image. In this example, when the immunocomplexes
were formed directly on the fiber array (i.e., a one-step assay
rather than a two-step, "capture-and-release", assay), non-specific
binding was typically observed to be approximately 2%. Therefore
the use of a two-step, "capture-and-release" assay, according to
this embodiment, had a dramatic effect on lowering NSB resulting in
greater sensitivity to low concentrations of analytes. This benefit
of the method "capture-and-release" may be attributed to a number
of factors, including, selective release by the dissociating agent
of immunocomplexes over non-specifically bound molecules and
advantageous surface chemistries of the first capture
components.
[0266] FIG. 27 shows results for the detection of TNF-alpha using
the method described in Example 11. In this particular experiment,
detection limits in single digit femtomolar ranges could be
obtained as compared to 10.sup.3 femtomolar (e.g. 1250 fM) using
the substantially the same reagents on a traditional ELISA plate
assay (see FIG. 28, Maxisorb ELISA 96well plate, same reagents and
incubation times as used in comparative assay, detection on a Tecan
M.sub.200 plate reader). FIG. 29 shows a similar assay performed
according to an embodiment of the inventive methods for the
detection of TNF-alpha where the beads were modified with capture
components comprising anti-TNF Ab stabilized with bovine serum
albumin. In this example, the capture beads were prepared by
reacting the beads with a 1:1 mixture of anti-TNF Ab and BSA,
instead of pure anti-TNF Ab. The theoretical limit of detection in
this assay was 8 fM.
[0267] FIG. 30 shows results for the immobilization of Fab'
fragments of an anti-TNF monoclonal antibody (MAB610) generated
using enzymatic degradation and purification as the capture
components immobilized on beads. The graph shows a comparison of
detection of TNF using MAB610 on beads versus Fab' of MAB610 on
beads where part of the immunocomplex comprising SbG was released
with 10 mM bME. In this embodiment, higher signals but also higher
NSB backgrounds were observed when using Fab' fragments.
[0268] In another experiment, the TNF molecules were captured on an
ELISA 96-well plate as a first substrate (Maxisorb, Nunc) as
opposed to beads. In this case, SbG of the immobilized complex was
released using bME and was detected on an optical fiber bundle.
FIG. 31 shows a graph of results obtained using the plate as the
first substrate. A substantially greater sensitivity was observed
as compared to results obtainable using traditional ELISA methods,
but less sensitivity was observed as compare to instances wherein
the analyte molecules were captured on beads.
[0269] FIG. 32 shows the results for a method of the present
invention, wherein one of the binding ligands of the assay
comprised a photocleavable linkage. In this example, the
immobilized complexes were formed on an ELISA 96-well plate as the
first substrate. The plate was exposed to UV radiation, and a
plurality of dissociated species was formed. The dissociated
species were then detected/quantified using methods described
herein. The graph depicts the plate reader fluorescence for three
samples: i) no target analyte present, ii) target analyte present
and the binding ligand does not comprise a photocleavable linker
and iii) target analyte present and the binding ligand comprises a
photocleavable linker.
Example 16
[0270] The following example describes a comparison between the
methods/systems of the present invention and a traditional ELISA
assay. Those of ordinary skill in the art will be familiar with the
techniques associated with an ELISA assay.
[0271] For the ELISA assay, a Maxisorb ELISA 96-well plate (Nunc)
was employed. The ELISA plate was functionalized with monoclonal
anti-TNF capture components. The substrate was then exposed to
varying concentrations of TNF-alpha. The substrate was additionally
exposed to a first binding ligand (polyclonal biotinylated anti-TNF
antibody) and a second binding ligand
(streptavidin-b-galactosidase). The conversion of substrate of
b-galactosidase was detected on Tecan M200 plate reader. In this
example, the limit of detection was approximately 1250 fM (see FIG.
33).
[0272] To compare with an assay of the present invention, magnetic
beads were provided which comprised monoclonal anti-TNF capture
components. The beads were exposed to varying concentrations of
TNF-alpha. The beads were additionally exposed to polyclonal
disulfide-containing biotinylated anti-TNF antibody and
streptavidin-b-galactosidase (SbG). At least a portion of the SbG
was released from the beads using 10 mM beta-mercaptoethanol. The
released complexes comprising SbG were captured on a fiber bundle
well array comprising biotin capture components. Each reaction
vessel was exposed to at least one precursor labeling agent
molecule comprising resorufin-beta-D-galactopyranoside (RDG) that
was converted to fluorescent resorufin upon exposure to SbG. The
presence of at least one labeling agent molecules formed via the
conversion of the precursor labeling agent molecule by the
b-galactosidase of the SbG in the reaction vessels was detected
using a fluorescence imaging microscope as described in Example 9.
In this embodiment, the limit of detection was less than 5 fM (see
FIG. 34).
[0273] While several embodiments of the present invention have been
described and illustrated herein, those of ordinary skill in the
art will readily envision a variety of other means and/or
structures for performing the functions and/or obtaining the
results and/or one or more of the advantages described herein, and
each of such variations and/or modifications is deemed to be within
the scope of the present invention. More generally, those skilled
in the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the teachings of the present invention
is/are used. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically described
and claimed. The present invention is directed to each individual
feature, system, article, material, kit, and/or method described
herein. In addition, any combination of two or more such features,
systems, articles, materials, kits, and/or methods, if such
features, systems, articles, materials, kits, and/or methods are
not mutually inconsistent, is included within the scope of the
present invention.
[0274] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0275] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," and the like are to
be understood to be open-ended, i.e., to mean including but not
limited to. Only the transitional phrases "consisting of" and
"consisting essentially of" shall be closed or semi-closed
transitional phrases, respectively.
* * * * *